http://2013.igem.org/wiki/index.php?title=Special:Contributions/R.Lizarralde&feed=atom&limit=50&target=R.Lizarralde&year=&month=2013.igem.org - User contributions [en]2024-03-29T07:57:18ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-10-25T01:59:05Z<p>R.Lizarralde: added an extra space below first video</p>
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<br><br />
Our project was less amenable to fancy 3D animations this year, so instead we decided to explore different kinds of animated visuals. The most successful project was MycoDraw, a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness.<br />
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Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration. We implemented it in Java and javascript, although we were unable to make the javascript run efficiently on certain browsers (notably Firefox). The code for the javascript can be found <a href="https://2013.igem.org/Team:Cornell/javascripts/mycodraw?action=raw">here</a>.<br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-10-25T01:58:33Z<p>R.Lizarralde: Removed mention of other animations, rewrote for mycodraw, replaced iframes with direct youtube links</p>
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<br><br />
Our project was less amenable to fancy 3D animations this year, so instead we decided to explore different kinds of animated visuals. The most successful project was MycoDraw, a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness.<br />
<br><br><br />
<iframe width="750" height="495" src="http://www.youtube.com/embed/n0LQmhcJTFw?html5=1&wmode=transparent" frameborder="0" allowfullscreen></iframe><br />
<br><br />
Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration. We implemented it in Java and javascript, although we were unable to make the javascript run efficiently on certain browsers (notably Firefox). The code for the javascript can be found <a href="https://2013.igem.org/Team:Cornell/javascripts/mycodraw?action=raw">here</a>.<br />
<br><br><br />
<iframe width="750" height="495" src="http://www.youtube.com/embed/bAtCY2tAyy4?html5=1&wmode=transparent" frameborder="0" allowfullscreen></iframe><br />
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<br><br />
Our project was less amenable to fancy 3D animations this year, so we took a different approach: trying to pack a lot of information in minimally designed, cartoon-style animation.<br />
<br><br><br />
<iframe width="755" height="520" frameborder="0" src="http://dl.dropboxusercontent.com/u/496608/animation.html" scrolling="no"></iframe><br />
<br><br />
However, we also worked on other visual displays, such as a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness. Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration.<br />
<br><br><br />
<iframe width="755" height="520" frameborder="0" src="http://dl.dropboxusercontent.com/u/496608/animation2.html" scrolling="no"></iframe><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/File:Styrofoamsmaller.jpgFile:Styrofoamsmaller.jpg2013-10-11T20:18:53Z<p>R.Lizarralde: uploaded a new version of &quot;File:Styrofoamsmaller.jpg&quot;</p>
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<div></div>R.Lizarraldehttp://2013.igem.org/File:CornellNovelty.jpgFile:CornellNovelty.jpg2013-10-11T20:16:01Z<p>R.Lizarralde: uploaded a new version of &quot;File:CornellNovelty.jpg&quot;</p>
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<div></div>R.Lizarraldehttp://2013.igem.org/File:Styrofoamsmaller.jpgFile:Styrofoamsmaller.jpg2013-10-11T20:15:16Z<p>R.Lizarralde: uploaded a new version of &quot;File:Styrofoamsmaller.jpg&quot;</p>
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<div></div>R.Lizarraldehttp://2013.igem.org/File:Ecovative_desing.jpgFile:Ecovative desing.jpg2013-10-09T19:12:04Z<p>R.Lizarralde: uploaded a new version of &quot;File:Ecovative desing.jpg&quot;</p>
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<div></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/notebook/protocolsTeam:Cornell/notebook/protocols2013-09-28T04:16:38Z<p>R.Lizarralde: </p>
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<a href="https://static.igem.org/mediawiki/2012/f/fb/Single_Digest.pdf" target="_blank">Single Digest</a><br />
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<a href="https://static.igem.org/mediawiki/2012/a/a7/T4_Ligase_Ligation.pdf" target="_blank">T4 Ligase Ligation</a><br />
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<a href="https://static.igem.org/mediawiki/2012/5/57/Making_Ecoli_Electrocompetent.pdf" target="_blank">Preparing Electrocompetent <i>E.coli</i></a><br />
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<h4>DNA Assessment</h4><br />
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<a href="https://static.igem.org/mediawiki/2012/0/03/Sequencing_Preparation.pdf" target="_blank">Preparation of DNA for Sequencing</a><br />
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<h4>Fungal Transformation</h4><br />
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<a href="https://static.igem.org/mediawiki/2013/f/f0/Transformation_of_Protoplasts.pdf" target="_blank">Transformation of Protoplasts</a><br />
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<h3 class="centered">January</h3><br />
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{{:Team:Cornell/templates/nbentry<br />
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|head=January 5th<br />
|text=Applications for new Cornell iGEM members were due today-- we've got 57!<br />
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|head=January 16th<br />
|text=After an eternity of reading and a gigantic spreadsheet of evaluations, we're finally ready to start interviewing applicants-- 36 of them!<br />
|author=Rafael<br />
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|head=January 25th<br />
|text=I'm really excited about having these new members on the team. There were many awesome interviews, but now we have to sit down and make hard decisions on who to take, as most of last year's members are graduating, so there's a limit to how many new members we can train. This is going to be a tough weekend.<br />
|author=Rafael<br />
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|head=January 27th<br />
|text=After several hours of discussion, we decided on our 21 new members! Congratulations, Tim Abbott, Hannah Ajmani, Eric Appel, Ryan Ashley, Nupur Bhatt, Arun Chakravorty, Rebecca Chew, Sharlene Dong, Sara Gregg, Alex Han, Eric Holmes, Daniel Leach, Oat Luengvarinkul, Jeffrey Ly, Ritvik Sarkar, Mac Sennett, Prashant Sharma, Olya Spassibojko, Yoshiko Toyoda, and Kyle Wheeler!<br />
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|text=We had our first full team meeting today with all the new members, introducing them to the iGEM competition, and did an icebreaker we call speed-taboodunit! For the next week, they have the assignment of analyzing a previous iGEM project (in groups), and explaining what the project was trying to accomplish, whether synthetic biology was a good approach for this, and reasons for their success or lack thereof.<br />
|tech=Speed-taboodunit is a mashup of speed-dating, taboo, and whodunit; the idea is that each person writes on a piece of paper something unusual about themselves, underlining the key words, then these pieces of paper are randomly redistributed. Everyone organizes in a speed-dating format, with two lines of people, and you have a minute to talk to them and try to find out if they're the person described on their piece of paper-- with the catch that they can't use any of the underlined words. People who successfully find their person leave the group, but otherwise after the minute everyone moves down the line. As for why we came up with this specific icebreaker, we wanted to hit two key requirements of icebreakers: they have to be fun, and they have to make specific accommodations for being shy or quiet. Because each person has the specific objective of finding their person, this helps them overcome the shyness, and as for the fun, everyone loves taboo!<br />
|author=Rafael<br />
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|head=February 7th<br />
|text=Our first social event tonight went well! Hopefully with a few more of these we'll have some great team comradery and feel more comfortable around each other. <br />
|author=Rafael<br />
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|head=February 9th<br />
|text=At our second team meeting we went over the past project analyses, as well as some biology review-- mostly molecular cloning. For the next week, we've assigned more past projects for them to outline in more detail.<br />
|author=Rafael<br />
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|head=February 16th<br />
|text=Today we went over literature review, went over the past project outlines, and gave a two-week problem-oriented brainstorming and project outline assignment. Now that each member has looked at several past projects and analyzed them, we're moving into brainstorming for this year's project and practicing the development of project pitches.<br />
|author=Rafael<br />
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|head=February 21th<br />
|text=We just started working on the wiki! Our first assignment: figuring out how wiki templates work. With templates the website would become much easier to edit, but for some reason HTML and wiki text don't seem to work very well together...<br />
|author=Nupur<br />
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|head=February 23th<br />
|text=For the second week of this assignment, we compiled the brainstormed ideas and had members pick which ones they wanted to explore further. We also had a movie night planned, but it fell through :(<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
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|head=February 28th<br />
|text=Finally got templates to work! The trick was ending HTML right before wiki text, and starting HTML again right afterwards. Not very elegant, but it works.<br />
|author=Nupur<br />
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|head=March 1st<br />
|text=We want to revamp the notebook page with a filter system that lets you sort by subteam. We set up the basic layout of the page, but writing the javascript is going to be another story.<br />
|tech=Check out our <html><a href=https://2012.igem.org/wiki/index.php?title=Team:Cornell/Sandbox/notebook&oldid=299789 target="_blank"> notebook</a></html> before anything worked!<br />
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|head=March 2nd<br />
|text=Groups over the previous week investigated phage therapy, piezoelectric bacteria, "sticky paper", logic gates / bistable switches, phytases, mycelia, and pressure-adaptive materials. We're keeping the results from those, but doing a second round of brainstorming over the next few weeks-- the idea is to allow the exploration of any ideas that arose while researching the previous round of ideas.<br />
|author=Rafael<br />
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|text=We just finished the javascript for filtering. Rafael also made cool subteam icons for the filter buttons.<br />
|author=Nupur<br />
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{{:Team:Cornell/templates/nbentry<br />
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|text=I made a template for notebook entries and added styling to each entry.<br />
|author=Nupur<br />
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{{:Team:Cornell/templates/nbentry<br />
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|head=March 9th<br />
|text=We compiled our second round of ideas, including topics like methanotrophs, a CRISPR knockout system, and suppressor tRNA inducible expression systems. We also took our first team picture, and probably the only one that will have all of last year's and this year's members together!<br />
|author=Rafael<br />
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|head=March 29rd<br />
|text=Spring break knocked out a couple of meetings, but it looks like some research was accomplished nonetheless! The phytase project idea is looking pretty promising-- it incorporates a lot of different disciplines, with stuff for our mechanical and electrical engineers to do, as well as modeling.<br />
|tech=A lot of iGEM teams we've looked at don't have much beyond the biology, so we really try hard to do interdisciplinary projects, as they provide a great opportunity for learning to work with different kinds of people.<br />
|author=Rafael<br />
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|head=April 6th<br />
|text=We've narrowed it down to four project ideas: smokestack reactors (capturing pollutants with bioreactors), incorporation of phytases into anaerobic digesters, mycelial materials, and using bacteria to address issues with artificial implantable kidneys.<br />
|author=Rafael<br />
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|type=wet<br />
|head=April 10th<br />
|text=We had a call today with Ecovative Design, a company that uses mushrooms to make a biodegradable styrofoam analogue-- our mycelial materials project idea would involve working with them to improve their product. It seems like our interests are aligned and that we should be able to work with them. Exciting! <br />
|author=Rafael<br />
}}<br />
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|head=April 13th<br />
|text=This meeting was dedicated to project pitches from each of the four ideas, followed by voting on which project we should pursue. Some of our graduate advisors also attended to provide a more seasoned perspective on the viability of each project. After some deliberation and spreadsheet magic, we decided on mycelial materials!<br />
|author=Rafael<br />
}}<br />
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|head=April 20th<br />
|text=Today we worked on team structure for the summer; Swati will be the overall team leader, with Mark and I coordinating wetlab, Olya taking meeting minutes, Mac running drylab, and Hannah will be managing human practices. Later on in the summer, we will also develop task groups. For the next week, we've also split into groups to research various aspects of the mycelial materials project.<br />
|author=Rafael<br />
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|head=April 26th<br />
|text=We've compiled initial research on carotenoid pigments, transformation protocols, DNA that may be useful for a basidiomycete toolkit, and a few other topics. Over the next week we'll meet with several professors on campus who work with fungi to see what resources and advice they can provide.<br />
|author=Rafael<br />
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|type=wet<br />
|head=May 4th<br />
|text=We met with Professor Gillian Turgeon, who does fungal transformation with Cochliobolus heterostrophus, an ascomycotic corn pathogen. We can get a few of the genes we've been looking for from her, yay! We also met with Professor Teresa Pawlowska, who directed us towards some other fungi and resources for fungi, as well as provided some background on fungal biology. As for the rest of the semester, we're letting everyone off the hook-- we don't want anyone to fail their exams because of iGEM!<br />
|author=Rafael<br />
}}<br />
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<div id="week1entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered" >Week 1</h3><br />
<h5 class="centered">(06/17 - 06/23)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 17th<br />
|text=The drylab team met briefly on June 17 to begin discussing some ideas. We went over some of the growing methods for Ganoderma lucidum, brainstormed some non-packaging related ideas for a fungal biomaterial, and discussed how we could test some of the physical characteristics of the final product. We thought it might be a good idea to test the thermal conductivity if it were to be used as a drinking cup or something. We also determined that people probably do not want to drink out of a fungus cup. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 18th<br />
|text=We started bootcamp today, doing our first transformations with kit plate biobricks.<br />
|tech=We transformed parts containing carotenoid pathway genes ''crtE'', ''crtI'', ''crtB'', and ''crtY'' (BBa_K523022, BBa_K539119, BBa_K145001), as well as the T7 promoter (both in pIVEX2.3d as well as BBa_I712074), the T7 polymerase gene (BBa_K145001), and the transformation efficiency kit part (BBa_J04450).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 19th<br />
|text=The second day of bootcamp had more activity-- we PCR-amplified a variety of parts to biobrick them and get ready for making composite parts later.<br />
|tech=From the plasmid pUCATPH (pAh13G), we amplified the ''trpC'' promoter (P''trpC'', a constitutive fungal promoter) and the ''trpC'' terminator (T''trpC''). From pNG (pAg13H), we amplified ''nptII'', the geneticin (a plant/fungal antibiotic) resistance gene.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 20th<br />
|text=I finally started moving pages to the 2013 wiki! It's looking very empty right now, but Rafael made a gif of our logo with his mycodraw program to use as a homepage placeholder. We also decided to add a technical details section to each of our notebook entries so that we can separate the jargon from daily summaries.<br />
|tech=Check out the <html><a href=https://static.igem.org/mediawiki/2013/2/21/CornellStillGrowing.gif target="_blank">gif</a></html> if it's not on our homepage anymore. Originally Rafael converted his mycodraw program from java to javascript (which took a VERY long time), but the script only worked on some browsers :( We had to settle for the gif instead.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 20th<br />
|text=On our third day of bootcamp, we taught new members how to run gels, do minipreps, and run digests, continuing our preparation of biobrick parts from the previous two days.<br />
|tech=We miniprepped the ''crtEIB'' (BBa_K523022), ''crtY'' (BBa_K539119), and T7 (BBa_I712074) biobricks. The gels confirmed our PCRs for P''trpC'', T''trpC'', and ''nptII'', which we then digested to insert into pSB1C3.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 21st<br />
|text=The javascript for toggling technical details took a bit longer than expected, but we finished it up today.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 21st<br />
|text=Continuing with bootcamp, we column-purified the digests of PCR products and gel-purified the digested pSB1C3 backbone, dephosphorylated, and ligated, resulting in our first batch of biobricks or pre-biobrick parts.<br />
|tech=P''trpC'', T''trpC'', and ''nptII'' were column-purified, digested pSB1C3 backbone was gel-purified and dephosphorylated, and each was ligated into the pSB1C3 backbone.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 22nd<br />
|text=I added a flyout to notebook entries authors with a picture, short description, and link to their bio on the team page.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 22nd<br />
|text=Our penultimate day of bootcamp was a light one-- we only transformed cells with the constructs we'd made over the past four days. <br />
|tech=We transformed T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) constructs into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 23rd<br />
|text=We concluded bootcamp with colony PCRs to test whether each of the constructs was present. Unfortunately, the gels had no bands, so we grew overnight cultures and will screen the minipreps instead.<br />
|tech=We used Q5 PCRs with the standard biobrick primers VF2 and VR to attempt to confirm the presence of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R).<br />
|author=Rafael<br />
}}<br />
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<div id="week2entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 2</h3><br />
<h5 class="centered">(06/24 - 06/30)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 24th<br />
|text=The drylab team met to update each other on some of the preliminary research. The main focus of this meeting was the development of a growing protocol. Sara found a professor that we could contact in the material science department for help regarding the testing of material properties. Rebecca and Manny looked into what different aspects of the fungi we could model, such as growth, decay, etc. Ritvik brainstormed some more ideas regarding the potential uses of this material, and came up with the idea of using it as insulation. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 24th<br />
|text=I started working on a hypertexting script that will automatically recognize words on a page and add a flyout with a description. We can use this to recognize names for the author flyouts I implemented yesterday, as well as other keywords such as plasmid names and protocols.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 24th<br />
|text=We miniprepped our three constructs. Then we went back to sleep.<br />
|tech=Minipreps of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 25th<br />
|text=I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 25th<br />
|text=We did another PCR from the miniprepped plasmids to confirm the right sizes of inserts, then sent them in for sequencing.<br />
|tech=The Q5 PCRs of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) showed bands of the correct size, so sequencing with VF2 and VR was submitted to make sure nothing strange had happened.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 26th<br />
|text=The script is now working on all browsers except for Internet Explorer.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 27th<br />
|text=We started cloning crt genes downsteam of the T7 promoter. Because the T7 promoter is too small to extract as a fragment, we're placing crt genes downstream, then transferring the promoter+gene fragments back to pSB1C3. Today we digested ''crtY'' and the T7 promoter plasmid, but the digestion of ''crtY'' failed. <br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). However, the gel showed only one band for each (what one would expect for pAK13D, but pC13B should have had two bands).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 28th<br />
|text=We redid the digestion of ''crtY'' and the T7 promoter plasmid from yesterday, apparently successfully this time. We also started liquid cultures of pBARGPE1, a plasmid we got from the Fungal Genetics Stock Center.<br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). The gel this time had two bands at the correct locations for pC13B, so we're hoping it's a success this time. We also started overnight LB cultures of pAb13T (pBARGPE1).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 29th<br />
|text=Crossing our fingers, we gel extracted and ligated the ostensibly successful digests of ''crtY'' and the T7 promoter plasmid, and then transformed. We also digested ''crtI'' and ''crtB'', other carotenoid genes that we will also be putting under the T7 promoter, and miniprepped the pBARGPE1 that had been grown up last night.<br />
|tech=If successful, the ligation will form pAK13AD. pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI for insertion into pAK13D, and separated on a gel. pAb13T (pBARGPE1) was miniprepped and quantified by nanodrop. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 30th<br />
|text=Unfortunately, there were no colonies on the transformation plate for ''T7'' + ''crtY'', so we redid the digestions for those. We also digested ''TtrpC'' and ''nptII'' for the eventual cloning of a ''PtrpC''+''nptII''+''TtrpC'' construct (we have to put the parts together in reverse because ''nptII'' contains a PstI site, which we will be trying to remove as well). Gel extractions of digested ''crtI'' and ''crtB'' were also empty (insignificant concentrations), so these digestions were also redone, and all were run on a gel, which looked mostly okay.<br />
|tech=pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (''TtrpC'') with EcoRI and XbaI, and pCg13R (''nptII'') with EcoRI and SpeI, and all were separated on a gel; ''crtI'' looked faint, and pAK13D was smeared, but all fragments were cut out for purification tomorrow.<br />
|author=Rafael<br />
}}<br />
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<h3 class="centered">Week 3</h3><br />
<h5 class="centered">(07/01 - 07/07)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 1st<br />
|text=We finished the hypertexting script and fixed some miscellaneous problems. I think we can call this a wrap. The notebook page is officially done!<br />
|tech=Now we just have to start filling out entries...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 1st<br />
|text= Two of our team members participated in SILS Skills Night on June 20th.<br />
|tech= Price of one 12 oz Styrofoam cup: 4.09 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 1st<br />
|text=Yesterday's digestions were gel extracted, ligated, and transformed. Hopefully it worked this time!<br />
|tech=Digested pAK13D was dephosphorylated, then ''crtI'' , ''crtB'', and ''crtY'' were ligated with pAK13D to put them under the T7 promoter, hopefully producing pAK13AB, pAK13AC, and pAK13AD, respectively. pCg13R was also ligated with pC13Q, hopefully producing pCg13U. All four constructs were transformed into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 2nd<br />
|text=There were no transformants, again :(. On the other hand, we received our tube of <i>Ganoderma Lucidum</i> from Dr. David Hibbett today! Once more with feeling, we redid the four digestion-ligations for ''crtI'', ''crtB'', ''crtY'', and ''nptII''. We also amplified ''hph'', the hygromycin resistance gene, from one of the plasmids we got from Dr. Turgeon's lab, as well as pSB1C3 backbone, and we will use Gibson assembly to put them together (as ''hph'' has both EcoRI and PstI sites)-- the gels looked good for both of these. Finally, we submitted pAb13T (pBARGPE1) for sequencing to determine the sequences of the ''Aspergillus nidulans'' ''gpdA'' promoter and the ''bar'' gene, which confers phosphonothricin resistance.<br />
|tech=''hph'' was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 3rd<br />
|text=All transformations failed yet again, and were repeated once more. Four tears shed for the lost. ''hph'' was Gibson-assembled, and we ran a Q5 PCR of the ''bar'' gene, under the assumption it matches other published ''bar'' sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on ''nptII'' (pCg13R) to remove its internal PstI site.<br />
|tech=:( ;_; T_T D: (tears for the transformations lost). The ''bar'' PCR and pC13F were digested with EcoRI and PstI, and ligated to hopefully form pCp13X. Site-directed mutagenesis was performed both with and without DpnI to digest template DNA. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 4th<br />
|text=The previous days ligations were all desalted and transformed, yet again.<br />
|tech=The previous days ligations were all desalted and transformed, yet again, into E. Coli and plated, including the non-GC trials of ''nptII'' site-directed mutagenesis. Digestions on ''bar'' and pSB1C3 were purified and run on a gel. They appeared successful, and pSB1C3 was gel-extracted for ligation with the PCR-cleanup of ''bar''. The digests of pSB1C3 and ''bar'' were quantified and ligated for submission as a Biobrick. Finally, liquid broth cultures of the hygromycin Gibson transformants were created.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 5th<br />
|text= All of yesterdays transformations failed, in addition to the ''hph'' Gibson :-( The ligase was tested by ligating DNA ladder: All were discovered to be non-functional. Why life? Why?<br />
|tech= The site directed mutagenesis cultures of ''nptII'' were put into liquid cultures from plates. One liquid culture of a transformant of ''crtY'' in pAK13D was also made, along with a reference plate. A ligation of ''bar'' in pSB1C3 was desalted and transformed. Only one of the Gibson assemblies appeared successful, failing to express rfp, and a glycerol stock was made of that culture. After further incubation, the successful culture was miniprepped and PCR'ed.When run on a gel, that culture appeared ineffective.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 5th<br />
|text=Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 6th<br />
|text=The ''crtY'' ligation was run on a gel and shown to be unsuccessful. The ''hph'' PCR also came up blank, so the Gibson was also unsuccessful. On the bright side, a digestion screen on ''nptII'' shows that site-directed mutagenesis on that site most likely worked!<br />
|tech=Colonies of ''nptII'', ''nptII'' digested with DpnI, ''hph'', and ''crtY'' were miniprepped. ''nptII''+ DpnI sample 4 was accidentally pipetted into the sample 3 column, so contamination is possible. Concentrations were good. A digest screen was performed on ''nptII'' using PstI, and ''crtY'' was digested with EcoRI and PstI. Fragments of ''nptII'' digested with DpnI ~2.5kb and linearized mutated ~3kb. The ''nptII'' digested with DpnI appeared successful, while the undigested samples failed. Gels of ''hph'', and ''crtY'' were also run, showing that the ''crtY'' ligations failed, as well as the ''hph'' Gibson.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 7th<br />
|text=A pSB1C3 backbone was prepared for Gibson assembly and gel extracted; however, we later were told by a representative from New England Biolabs that gel extraction might mess up a Gibson assembly, rendering todays extractions useless.<br />
|tech=A Miniprep was run on three ''bar'' cultures in preparation for later ligation into pSB1C3. These were then run on a gel along with Gibson-ready pSB1C3. Although loading dye bubbling out of the wells made it appear as though no results would come of the gel, the gel was successful. ''bar'' was confirmed and a gel extraction was performed on pSB1C3 and frozen.<br />
|author=Kyle<br />
}}<br />
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<div id="week4entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 4</h3><br />
<h5 class="centered">(07/08 - 07/14)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 8th<br />
|text=Several constructs were ligated. The site-directed mutagenesis of ''nptII'' in pCg13S was ligated with ''TtrpC''. Assembled ''bar'' in pSB1C3, submitted for sequencing. The Gibson of ''hph'' into pSB1C3 was rerun. An experimental method of ligating ''crtY'' into pAK13D upstream of the T7 promoter was tried as an alternative to gel purification and PCR purification, since we lack the primers to PCR and gel purification interferes with ligation.<br />
|tech=Digested ''nptII'' in pCg13S with EcoRI+XbaI, and TtrpC with EcoRI+SpeI. The ''crtY'' in pSB1C3 was digested four different ways: One with just XbaI & PstI; one with XbaI, PstI, and SacI; one with XbaI, PstI, and ApaLI; and one with XbaI, PstI, SacI, and ApaLI. Although unlikely, this method is intended to prevent re-ligation of the backbone of ''crtY'' during digestion. More ''crtY'', ''crtI'', ''crtB'' was inoculated in liquid cultures for future miniprepping.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 9th<br />
|text=All yesterdays ligations were dialyzed and plated. The Gibson of ''hph'' into pSB1C3 grew red colonies, showing it was unsuccessful, so the procedure was performed again. The ''bar'' ligation came back successful!<br />
|tech=<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 10th<br />
|text=The ''nptII'' ligation with ''TtrpC'' grew colonies was unsuccessful. In other news, much lab organization was done today!<br />
|tech=The ''nptII''+''TtrpC'' ligation was checked by doing a colony PCR on the ligation colonies and a subsequent gel. Possible gel outcomes were plasmid self ligation (200bp) in which ''TtrpC'' was randomly cut out of pC13Q, 800bp if the plasmid has only ''TtrpC'', ~1kb indicating ''nptII'' noise from PCR template, and ~1.7 kb indicating successful construct formulation. The gel showed two bars at ~800bp, indicating a failed ligation. The third Gibson attempt of ''hph'' into pSB1C3 was also plated, and a 10mL culture was made of the successful ''nptII'' DpnI mutagenesis plate, evidently for future great justice.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 10th<br />
|text= Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 11th<br />
|text=After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop. <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 11th<br />
|text=Digest screens of previous ''crtY'' ligations showed promising results. Gibson cloning was continued, and further cloning of ''nptIIBB'', to place it in front of PtrpC, was prepared.<br />
|tech=Minipreps were made from the ''crtY'' plates, from which a digest screen was prepared using NotI to see if the ligation worked. The digestions were run on a gel. Samples with XbaI, PstI, and SpeI; XbaI, PstI, and ApaLI numbers 2 and 3; and XbaI, PstI, SpeI, and ApaLI numbers 2 and 3 all worked. A miniprep of ''nptIIBB'' was performed. The concentration was extremely low, so an overnight culture was made from the glycerol stock to be miniprepped tomorrow. Another transformation was done using remaining previous minipreps of ''nptII BB''. Cultures were made of the third Gibson attempt, and a colony PCR performed. When run on a gel, the non-GC fragments were too small, but GC colonies number 2 and 5 were good, at 1kb, and number 4 had a band at 1kb with another smaller fragment. Finally, ''nptII BB'' was digested with XbaI and PstI, then column purified. PtrpC was digested with SpeI and PstI and gel purified.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 12th<br />
|text=Cloning with ''nptIIBB'' and PtrpC was continued, as well as cloning with the Gibson Assembly method.<br />
|tech=In the morning, the digestions of ''nptIIBB'' and PtrpC were quantified. A miniprep of ''nptIIBB'' transformants was performed to replenish DNA, and another glycerol stock of ''nptII BB'' was made from a leftover culture. The GC colonies 2, 4, and 5 from yesterdays gel were prepared for sequencing. A ligation was performed on ''nptIIBB'' and PtrpC. More cultures of the successful ''crtY'' ligations were made. CYM medium was created, and ''PC13Z'' was transformed from a kit plate.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 13th<br />
|text=Today we worked with fungi! We applied different amounts of antibiotic to G. lucidum, to test the sensitivity of our strain of fungi. In addition, cloning of pCg13Y was nearly completed. <br />
|tech=The ligation of ''nptIIBB'' with PtrpC was desalted, transformed into E. coli and plated. Antibiotic stocks of geneticin and phosphinothricin were made at 100x concentration. <i>G. lucidum</i> cultures were inoculated with varying levels of antibiotic to test the strain's sensitivity for future selection purposes.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 14th<br />
|text=Cloning of ''nptIIBB'' in front of PtrpC was unsuccessful. Plasmids pC13z and pAK13AD were prepped for cloning.<br />
|tech= Colony PCRs and cultures were completed to determine the success of cloning ''nptIIBB'' downstream of PtrpC. Unfortunately, a gel revealed that the dna fragments appeared at 0.6kb, shorter than the expected 1.4kb of a successful ligation. pC13Z and pAK13AD were miniprepped, but had to be thrown out due to contamination of our EB. New cultures were made to be miniprepped again. A glycerol stock of pC13Z was also made.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 14th<br />
|text=This week, drylab split up so members could focus on their strengths. Rebecca and Sara began designing our feedback controller, with some much appreciated advice from the previous drylab team leader, Dan. Ritvik, Manny, and Mac began coming up with design considerations for the incubation chamber. We were most concerned with size; too small and it wouldn't be useful, too big and it would be difficult to control any variables. We eventually decided that a 1'x1'x2' chamber would probably be a good compromise. Rebecca, Sara, Ritvik, and Mac also attended machine shop training this week. In order to complete our training, we each had to construct a small aluminum lamp using a milling machine and a lathe. Fortunately, everyone emerged from the training session one lamp richer, and with a full set of fingers on each hand.<br />
|author=Mac <br />
}}<br />
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<div id="week5entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 5</h3><br />
<h5 class="centered">(07/15 - 07/21)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 15th<br />
|text=Rebecca looked into the components of the temperature feedback control, namely, the temperature sensor, the microcontroller, and the heating element. Put in an order for the temperature sensor, microcontroller (an Arduino Fio), and Xbee ports for wireless communications.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 15th<br />
|text=Gibson ''hph'' was submitted for sequencing, and ALL of the cloning was performed.<br />
<br />
|tech=Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. To begin, the Gibson ''hph'' was submitted for sequencing, and pAK13AD and pC13Z were miniprepped. A PCR of pC13Z, pC13F, pCP13X, pC13M, and pC13E was then run with primers 31 and 32, the standard pSB1C3 primers. A gel was run to check the PCRs. All the PCRs looked successful except pC13M and pC13E, which had nonspecific bands around 1kb. A new technique was used on a few of the digests involving a restriction cocktail of enzymes to cut the DNA. Plasmid pAK13AD was digested with EcoRI, SpeI, SacI, and ClaI so that it could be placed in pSB1C3. We used SpeI instead of PstI due to the small distance between the PstI and the SacI cut sites. Plasmid pC13F was digested with EcoRI, SpeI, and KpnI so pAK13AD could be placed in it. Plasmid pAK13D was digested with SpeI and PstI so ''crtI'' and ''crtB'' could be placed in it. All the digests were then column purified rather than gel purified--a move which has improved our ligation efficiencies. The purifications were quantified. Cultures were made for pC13P and pC13K and, finally, pAK13AD was dephosphorylated then ligated into pC13F (pC13AH).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 16th<br />
|text=One iGEM member spoke at a panel for pre-freshmen biology students interested in pursuing research positions in college (PSP).<br />
|tech= Price of one 12 oz Styrofoam cup: 4.95 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 16th<br />
|text=The Gibson sequencing came back positive! We then digested pC13X, pC13F, pC13Z and pC13P, and ligated pAK13D to inserts pC13Z, pC13X and pC13F, and pC13P to inserts to inserts pC13Z, pC13X and pC13F. We ran a PCR for pC13M, pC13E and pC13K. Sadly pC13K did not work… Transformation was attempted for pC13AI and pA13AJ but they both failed--we suspected that the cuvettes weren't cleaned properly. <br />
<br />
|tech=Sequencing results came back positive for the Gibson cloning of hph, so a culture of Gibson colony 5 was made. pC13AH was transformed and plated, and pC13K were miniprepped. pC13X (SpeI/PstI), pC13F (SpeI/PstI), pC13Z (SpeI/PstI) were purified and digested along with pC13P (XbaI/PstI) [also dephosphorylated], then all were column purified. Reran a PCR of pC13M and pC13E, and one of pC13K with primers 31/32. All PCRs were cleaned, then run on a gel along with pBARGPE1 which had been digested with NotI today. Results: pC13E had a 3kb fragment (good), pC13K had a .9kb fragment (should be 1.7-1.8kb), pC13M had a 1.2-1.5kb fragment (good), pBARGPE1 had a 4kb fragment (super-coiled) and pBARGPE1 with NotI had a 5-6kb fragment (has a NotI site-good). From these results it can be concluded that everything worked except pC13K. Ligations of pAK13D to inserts pC13Z, pC13X, and pC13F along with vector pC13P to inserts pC13Z, pC13X, and pC13F were performed. Also, overnight digestions of pC13M, pC13E, and pC13K with XbaI and PstI were set up. Transformation was attempted for pC13AI and pA13AJ, but the cuvettes weren't cleaned properly so it failed. Finally, our dwindling supplies prompted the ordering of more SpeI and membrane filters. <br />
|author=Danielle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 17th<br />
|text=We reran a PCR for pC13K and the band was as expected on the gel! pC13M was ligated to pC13D, and pC13E was ligated to pC13P. The resulting constructs were used to transform ''E. Coli'' cultures. The following transformations were also done: ''pT7'' + ''GFP'', PtrpC + ''GFP'', ''pT7'' + ''BAR'', PtrpC + ''BAR''. A colony PCR was run for pC13AH.<br />
<br />
|tech= A glycerol stock of pCh13V (''hph'' Gibson) was created, then miniprepped for later site directed mutagenesis of ''hph'' (which has both E and P cut sites). Digestions of pC13M (crtB) and pC13E (T7 polymerase) were column purified and quantified. The PCR of pC13K with GC and non-GC trials was redone, since the fragment was of the incorrect length during gel imaging yesterday - goal is to ligate to pAK13D. Following the PCR, a gel was run which showed no bands. Perhaps the polymerase was forgotten? Another PCR of pC13K was performed and run on gel. A band showed at exactly 1.8kb as expected. pC13M was ligated to pAK13D, placing crtB downstream of the T7 promoter. pC13E was ligated to pC13P, placing the ''T7'' polymerase gene downstream of PtrpC (a native fungal promoter) - this is the first part of our genetic circuit with the second being the ''T7'' promoter upstream of various genes (GFP and RFP for diagnostic purposes - to quantify promoter functionality and the carotenoid genes for proof of concept). Plates of pC13AH (T7 + crtY) had many colonies, so they were restreaked, and colony PCRs were run while cultures were made of some of the colonies. A gel of the PCRs was run (image name was pC13AHcolonypcr7-17-13); gel was inconclusive (correct band was accompanied by strong incorrect band at 1kb). Will test again using digest screen of minipreps tomorrow. Heat shock transformed ''pT7'' + ''GFP'' (pAK13D + pC13Z), PtrpC + ''GFP'' (pC13P + pC13Z), ''pT7'' + ''BAR'' (pAK13D + pCp13X), PtrpC + ''BAR'' (pC13P + pCp13X) ligations into chemically competent E. coli. Heat shock transformed pC13AI (lox site), pA13AJ (lox site), pC13AK (mRFP coding sequence), and pK13AL (mRFP coding sequence) from Kit Plates 3 and 5 into chemically competent E. coli. Heat shock transformed today's earlier ligations: pAK13D + pC13M, and pC13P + pC13E and plated all transformations in accordance with the selection marker on the plasmid.<br />
|author=Danielle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 18th<br />
|text=The restriction cocktail method of cloning worked! Cloning of pC13AH (''T7'' + ''crtY'') was shown to work using this method. We are also continuing our cloning of the ''crtI'' and ''crtB'' pathways. <br />
<br />
|tech=All 18 (yes, 18!) of the ''pC13AH'' colonies that had been screened by colony PCR yesterday were miniprepped. We digested the miniprepped colonies of pC13AH clones (T7 promoter with crtY in pSB1C3) with ''NotI'' to see if the restriction cocktail method of cloning worked. On the gel that was subsequently run, bands were expected at ~2.1 for pSB1C3 and ~1.2 for ''crtY''+''pT7''. The restriction cocktail method worked! The bands appeared at the correct lengths on the gel for ''pC13AH''. There was not enough miniprepped DNA for pC13AH to send for sequencing, so the colonies were cultured again for another miniprep. None of the ligations into pAK13D (pSB1AK8 + PT7) worked -- pC13Z (''GFP''), pC13M (''crtB''), and pCp13X (''bar'') all failed => try cloning only using restriction cocktail method, instead of attempting PCR with VF2 & VR. Digested pC13Z, pC13M, pCp13X, pC13K (''crtI'') with XbaI and PstI in addition to ApaLI and SacI for later ligation with pAK13. pAK13D had been previously digested with SpeI and PstI, column purified, and dephosphorylated for insert ligation. More pAK13D was grown to acquire more DNA to be miniprepped tomorrow. Nupur performed colony PCR and made cultures of transformants from yesterday. All transformations appeared to have very low efficiency--perhaps an issue with heat shock stocks? Transformations of ''AI-AL'' (mRFP cds and lox site parts) all yielded colonies, though at really low efficiencies compared to most transformations off of kit plates. These were screened by colony PCR to verify. Ligations into pC13P all yielded colonies, but at very low efficiencies. These were pC13E (T7 pol), pCp13X (bar), and pC13Z. All were screened by colony PCR as well. Taq was used to perform 5 colony PCRs, then all colony PCRs were run on a gel. Also, pCg13S (nptIIBB)was digested with XbaI, PstI, ApaLI, and SacI for later ligation to pC13P, which had been previously digested with SpeI and PstI, column purified, and dephosphorylated. Overnight ligations of pC13Z, pC13M, pCp13X, pC13K, pCg13S with vector pAK13D were set up, along with overnight ligations of pC13K and pCg13S in vector pC13P. <br />
|author=Danielle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 19th<br />
|text=Sent pC13AH in for sequencing, and checked several plasmids for the presence of the correct genes using digestion screens and gel analysis.<br />
|tech=Made glycerol stocks of pC13AT, pC13AY, pC13AV, pC13AI, pA13AJ, pC13AK, pK13AL, and pAK13D. Miniprepped pC13AH cultures and sent in for sequencing. Ran a digest screen on pC13AI, pA13AJ, and pC13AL using NotI. Reran a PCR of pC13AV and pC13AT with Q5 polymerase. Attempted transformation of yesterdays ligations, with success only on pAK13AN and pAK13AD. A gel was run of pC13AL, pC13AJ, pC13AI (all digested w/ NotI), as well as the PCR of pC13AV and pC13AT. Plasmid pC13AT appeared to have worked, as well as pC13AI and pC13AJ, which had strong bands in the ~2kb range and weak bands in the ~1.5kb range, about the size of the whole plasmid.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 20th<br />
|text=Plasmid pC13AY was prepared for sequencing, and several transformations using heat shock were performed.<br />
|tech=Plasmid pC13AY was prepared for sequencing. Transformed pC13BD, pCg13AY, pAK13AC, pAK13BB, pAK13AB using heat shock. Recovered for ~1hr 10min, then plated on ampicillin plates made by spreading antibiotic on a sterile plate. More ampicillin plates were then created from scratch.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 21st<br />
|text=Made cultures of the previous days transformants, and ran an inconclusive digest screen of pAK13AN and pAK13AD. A slow day.<br />
|tech=Miniprepped pAK13AN, pAK13AD and ran a digest screen using NotI. The gel was inconclusive. The previous days transformants (pC13BD, pCg13AY, pAK13AC, pAK13BB, and pAK13AB) were cultured.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 21st<br />
|text=This week, the fabrication group focused on what materials to construct our chamber from. At first we considered a single wall design, but this was eventually scrapped, as we were unable to find any material that would provide sufficient insulation at a reasonable thickness (and cost). After putting our heads together, we decided on a double wall with an insulating layer in between them. Mac began drawing up the chamber in Solidworks. <br />
|author=Mac <br />
}}<br />
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<div id="week6entry" class="nb-week""><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 6</h3><br />
<h5 class="centered">(07/22 - 07/28)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 22nd<br />
|text= Sent in pC13AH, pC13AY, and pC13AT for sequencing. In addition, the digest screens of all the pAK13D ligations were redone, along with the colony PCRs of the pC13P ligations. Transformed pC13AV. Attempted to dry mycelium to isolate genomic DNA.<br />
|tech= Cultures pAK13AB number 5, pCg13Y number 1, pAK13AC number 1, and pCg13Y number 3 from yesterday didn't grow for some reason. Ran a NotI digest screen of all the pAK13D ligations, as well as colony PCRs of the pC13P ligations. Plasmid pAK13AB numbers 1, 3, 4 and possibly pAK13BB number 4 looked like they worked on the gel. The gel of the colony PCRs looked weird, possibly because they were run on wrong extension time for a while. Going to rerun PCRs. We also re-ran a gel of the NotI digest screens from yesterday, pAK13AP and pAK13AN. All the pAK13AP colonies had blank colonies, but pAK13AN colony 1 had the correct bands. Sent pAK13AN colony 1 for sequencing but accidentally sent in the wrong primers. Primers for pSb1AK8 should have been used, but pSB13C primers were used instead. Transformed pC13AV ligation and pC13AL. The transformation arced, but the time constant was still 3.8 so the cells were plated anyway. Finally, 2 250mL solutions of cym agar were made and autoclaved. Began drying mycelium to isolate the genomic DNA.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 23rd<br />
|text= Miniprepped and made a glycerol stock of pAK13AN. Also retried the cloning for pAK13AP and pAK13AC and began cloning for pAK13AO. Plasmid pC13AH was sequence confirmed!<br />
|tech= The plate of pK13AL transformation had no colonies, so it was given more time to grow. The plate of pC13AV had a lawn, but also lots of small isolated colonies. Plate was re-streaked to get fewer colonies before culturing. In addition, miniprepped and made a glycerol stock of pAK13AN and re-ran a colony PCR of pC13BD and pCg13Y with Taq polymerase. Ran a gel to see if ligation worked, but the bands were too short. This may be due to using Taq instead of Q5, so the PCR was run again with Q5. Retried cloning pAK13AP and PAK13AC, digesting pAK13D with SpeI and PstI, and pC13X and pC13K with XbaI and PstI. Overnight ligations were created, after column purification and dephosphorylation. We will redo a digestion of pC13AK tomorrow because we digested with SpeI and PstI instead of XbaI and PstI. We began cloning of pC13AQ, pC13BC and pC13AF, digesting backbone and insert with EcoRI and PstI. We made more DH5alpha electrocompetent cells to try to make heat shock. To replenish, TAE and LB were made. <i>G. lucidum</i> finished drying.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 23rd<br />
|text=The parts have arrived and Rebecca configured them. Met up with Paras who was on last year's team to get a detailed explanation of how their project had worked as well.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 23rd<br />
|text=Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 24th<br />
|text= Ran a gel of the Q5 PCR of the pCg13Y and pC13BD. One sample of pC13BD looked good, so it was sent in for sequencing. Isolating <i>G. lucidum</i>'s DNA didn't work. Will try flash freezing the cells with liquid nitrogen next time. <br />
<br />
|tech= Ran a gel of yesterday's Q5 PCR of pCg13Y and pC13BD, but only one sample of the latter looked good and was sent in for sequencing. Isolating <i>G. lucidum</i> DNA did not work because it did not precipitate. Next time, we will freeze the cells with liquid nitrogen. Re-tried cloning of pC13AY, pC13AT, and pCg13Y. Plasmids pC13E, pC13Z and pCg13S were digested with XpaI, PstI, SacI, and ApaII, while a previous digest of pC13P was used. Subsequently, pC13E, pC13Z, and pCg13S were column purified and the plasmids left to ligate overnight. Tried to redo cloning of pC13AU and pAK13AO because pC13AK was digested with the wrong enzymes yesterday, but failed once again. Finally, pC13I was cloned and pC13BD and pC13Z were submitted for sequencing.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 25th<br />
|text=We tried the site directed mutagenesis of pCh13V to remove the Pst1 internal cut site. Looking at the gel, it looks like the site directed mutagenesis worked! In addition, we cloned <i>crtE</i> into pSB1C3 and performed a ton of transformations!<br />
<br />
|tech= After growing up more pC13AK, attempted site directed mutagenesis to get rid of the PstI internal cut site in pCh13V. After running a gel against an unmutated control, found that GC worked while non-GC didn't. Afterwards, both were digested with DpnI and column purified. Attempted to clone pC13I. Also transformed ALL of the constructs, including: pC13AK, pK13AL, pA13AM, pCp13Y + pAK13AD, pAK13AC, pAK13AO, pCg13Y, pC13AY, pC13AU, pC13AV, pC13AT, pC13AQ, pC13AF, pC13I(crtE in psB1C3), and pCH13V GC with PstI mutation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 26th<br />
|text= Sadly, all 20 transformants failed and the stocks are incompetent. We received new cuvettes to retry all of them. We submitted ''lox'' site for sequencing of pBARGPE1 and ran a PCR of pC13F to append Kpn1 and BamHI restriction sites to pSB1C3 for cloning of “PpelA”.<br />
<br />
|tech= Sequencing results showed that pC13BD had the correct reverse sequencing, but the forward sequencing is being re-run. In addition, all 20 transformations failed due to incompetent stocks. Miniprepped and made glycerol stocks of pC13AK for more DNA and made many new plates. Also submitted the <i>lox</i> site for sequencing and ran a PCR of pC13F to append Kpn1 and BamH1 restriction sites to PSB1C3 for cloning of <i>PpelA</i>. We also ran PCRs using primers 27 and 28 to amplify <i>A. nidulans</i> <i>PgpdA</i> out of pAb13T, and ran a PCR using primers 29 and 33 to amplify <i>crtE</i> + <i>rbs</i> for pC13J out of pC13A. When all was said and done, the gel showed that only the PCR of <i>A. nidulans</i> looked promising, so we cloned this into pC13F.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 27th<br />
|text= We retried the PCR of pC13F, ran 63 colony PCRs, and made 97 cultures!<br />
|tech= Prepped pC13BD for sequencing, and retried the PCR of pC13F to append KpnI and BamHI restriction sites to clone <i>PpelA</i>. Ran 63 colony PCRs and made 97 cultures of all the previous ligations/transformations from two days ago. Also ran gels of the colony PCRs, but forgot to run the pC13F ones.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 28th<br />
|text= We digested pC13F and miniprepped cultures of yesterday's successful colony PCRs. <br />
|tech= Ran PCRs of pC13F on a gel to check for <i>PpelA</i>. The Non-GC PCR products looked good, but the products in which we used the GC Enhancer did not work. Cleaned up the non-GC enhancer PCR of pC13F and digested it with Kpn1 and BamH1. Also digested pHNU with Kpn1, BamH1, and Sac1. Then the double digest products were ligated overnight. The cultures of pA13AM (they did not grow before) were also retried. Miniprepped the cultures from yesterday whose colony PCR gel screens looked promising. Prepped the colony PCRs for: pC13AQ number 1, pC13AV number 5, pC13V PstI mutant numbers 2 and 3, pC13AF 4.3 and 4.5, and pCg13Y number 1. Also retried the questionable colony PCRs with Q5 off of the minipreps: pC13AY numbers 1-3, pC13AU numbers 1 and 5, pC13I numbers 4 and 5, pC13AT numbers 2 and 3, pAK13BC numbers 1 and 5, and pC13AF numbers 3.2 and 3.3. We digest screened all non-chloramphenicol minipreps using NotI-HF for pAK13AC numbers 1-5, and pAK13AO from July 23rd numbers 1-5. For everything else EcoRI and PstI was used.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 28th<br />
|text=This week, the fabrication group finalized the incubator design and ordered the construction materials! We decided to construct the outer and inner layers out of HDPE plastic in the rather lab appropriate color of white. For our insulation, we decided to use high density polystyrene. After comparing the K-factors for various types of insulation, we determined that this material gave us the most bang for our buck, so to speak. <br />
|author=Mac <br />
}}<br />
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<div id="week7entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 7</h3><br />
<h5 class="centered">(07/29 - 08/04)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 29th<br />
|text=Reran many PCRs and other procedures.<br />
|tech=Ran a gel of the questionable PCRs from yesterday. Plasmids pC13AY number 1, pC13AU number 5, pC13I number 5, pAK13BC number 5, and pC13AF number 3.2 looked good, but the gel was smeary so will re-run tomorrow. From the digest screens, pK13AL numbers 1-3, pAK13AP numbers 1-3 and 5, and pAK13AC number 1 looked good on a gel. The site-directed mutagenesis of pCh13V was continued, with a PCR of pCh13V run to mutate the EcoRI site. Sample number 2 of the non-GC samples appeared to have worked when run on a gel, so a DpnI digest and transformation were performed. Miniprepped pA13AM and ran a colony PCR, revealing that it was not in pSB1C3 as expected and thus was thrown out. Ran a digest screen of pA13AM with NotI to be checked on a gel tomorrow. <i>PpelA</i> ligated into pSB1C3 and transformed. Finally, after a long day, retried the PCR of pC13A to obtain <i>crtE</i>.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 30th<br />
|text=Yesterdays transformations all failed, and other mishaps. But fungal DNA extraction was successful!<br />
|tech=Submitted pC13BD, pC13AT, pC13AV, pCh13V mutant and pAK13AB for forward sequencing. Yesterdays transformations failed. A digest screen of pC13A and pC13AM revealed that the primers for pC13A were not functioning, and still no pC13J. Reran colony PCRs from gel yesterday. Digested pC13AL with XbaI, PstI-HF, ClaI, and SacI-HF and ligated into pAK13D, as well as digested pAK13AP and pAK13AC with XbaI and PstI-HF and ligated both into pSB1C3. Re-ran the ligation of <i>PpelA</i> into pSB1C3, as well as the mutagenesis PCR of pCh13V. Fungal DNA was successfully extracted!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 31st<br />
|text=Successful returns from sequencing of pC13BD, pCg13Y, pAK13AN and pC13V PstI mutant. In other news, the PCRs of <i>Ganoderma</i> DNA looked good!!<br />
|tech=Column purified pC13W DpnI digests: 3 GC and 3 nGC transformed and plated. Dephosphorylated pA13AM and ligated with pC13E. Transformed ligations from yesterday: pAK13AO, pC13AS number 3, pC13AG number 1, and pC13BF. Successful returns from sequencing of pC13BD, pCg13Y, pAK13AN and pC13V PstI mutant. Transformed and plated <i>Aspergillus</i> <i>Pgpd</i> in pC13F GC and non-GC.<br />
<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 31st<br />
|text=Sara and Rebecca met up with our advisor Dr Bruce Land and he provided lots of helpful advice with regard to the design considerations (where the heater should be in our incubation chamber, convection currents, type of heating plate to use). He also suggested that we use a proportional-integral-derivative controller (PID controller) to regulate the heating.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 1st<br />
|text= Ligation of the ''PpelA promoter'', hygromycin resistance, and ''T7 promoter'' in front of the herbicide-resistance bar gene worked. We continued cloning of our other constructs and repeated PCRs of <i>G. lucidum</i> GPD homology regions.<br />
|tech= We did colony PCRs with biobrick primers 31 and 32. We also made cultures of yesterday's transformations: pC13D+pK13AL, pC13D+pCp13X #3 in pSB1C3, pC13D+pC13K #1 in pSB1C3, pC13W with GC #3, pC13W no GC #3, and ''PpelA'' in pSB1C3. A gel was run to see if the ligations were successful. pC13D+pC13X, PpelA, pC13W GC 3.1 and 3.5, and pC13W nGC 3.1 all looked like they worked. The pA13AM+pC13E ligation from yesterday was transformed and plated. <i>G. lucidum</i> GPD homology regions were cloned, column purified, and digested with EcoRI and Pst. pC13F was also digested with EcoRI and Pst. Unfortunately, digestions of the homology regions yielded no DNA, but digestion of pC13F did yield some and thus was dephosphorylated. There was not enough template from the PCR cleanup present for homology regions, so PCRs were redone for each homology region.<br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 1st<br />
|text= Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 2nd<br />
|text= We continued working with the PCRs of the GPD homology regions and the successful ligations of the previous day's PpelA promoter, hygromycin resistance, and T7 promoter in front of the herbicide-resistance bar gene, as well as T7 polymerase behind a constitutive promoter and <i>Aspergillus</i> promoter. <br />
|tech= We cloned <i>G. Lucidum</i>GPD homology regions (pC13BJ, pc13BK, pC13BL, pC13BM). Yesterday's PCR reruns were run on a gel to make sure the right regions were amplified. All have correct bands! We column purified the reruns and digested with EcoRI and PstI. We also digested more ''pC13F'' with EcoRI and PstI and dephosphorylated the vector. We purified and ligated the reruns with pC13F. The cultures that looked good from yesterday's colony PCRs were miniprepped. We then made cultures and did a colony PCR of A.Pgpd in pSB1C3 GC from July 31st. A gel of the colony PCRs was run - colonies 1, 3, 4, and 5 looked successful and had the correct band insert length. A transformation of pA13AM+pC13E ligation did not work since the plate was contaminated. Sequencing of pC13AS #1, 3, 5 and pCh13W (hph BB) GC 3.1, 3.5, nGC 3.1 was prepared. An overnight culture of PpelA #2,3,5 was set up for miniprepping tomorrow and sequencing. We transformed pC13P+pC13E #3 and plated for cultures tomorrow, which we will then miniprep and sequence.<br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 2nd<br />
|text= Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 3rd<br />
|text= We continued cloning of T7 polymerase behind a constitutive promoter, transforming the GPD homology regions, and biobricking the carotenoid and geneticin resistance genes. <br />
|tech= We inoculated five colonies of pC13AY and five colonies of pA13AM+pC13E. We performed colony PCR of pC13AY transformants. A gel was run, but we realized that the PCR failed due to the use of Taq instead of Q5 and a 1 min shorter cycling time than the protocol required. We grew a culture of pC13P for miniprepping, digesting with Spe and PstI, and ligation with pC13Z and pK13AL. A digest screen of pAK13AO minipreps was performed - they were incorrect. pC13M, pC13K, pC13Z, pK13AL, and pCg13S were digested with Xba, Pst, Sac, ApaLI. An overnight ligation of pC13M + pAK13D , pCg13S + pAK13D, pC13K + pAK13D was performed. PpelA and A.Pgpd cultures were miniprepped. pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BM, and pC13F+pC13BL were all electroporated and plated. <br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 4th<br />
|text= We grew cultures of the GPD homology region transformations and transformed yesterday's carotenoid genes and geneticin resistance constructs.<br />
|tech= We miniprepped pC13AY, pC13AM+pC13E colonies, and pC13P. The minipreps were stored in the fridge, and glycerol stocks were made in case one of them works. We grew overnight cultures of five colonies from pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BM, and pC13F+pC13BL from yesterday's transformations. <br />
We transformed and plated pC13M + pAK13D, pCg13S +pAK13D, and pC13K +pAK13D. More LB agar and ampicillin plates were also made today.<br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 4th<br />
|text=Mac went to visit Ecovative Design with Swati, Rafael, Alex, and Hannah. We were able to meet with Gavin McIntyre, one of the founders of the company, who was kind enough to give us a tour of their facilities. He also gave our wetlab members some pointers for our own project. In other news, we are still waiting for our materials to arrive. <br />
|author=Mac <br />
}} <br />
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<div id="week8entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 8</h3><br />
<h5 class="centered">(08/05 - 08/11)</h5><br />
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<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 5th<br />
|text= Our previous construction of pC13AY failed. We submitted pC13BF and pC13BG for sequencing. We ran a colony PCR for pAK13AB, pAK13AC and pAK13BB. A few pAK13AC and pAK13BB colonies were successful. <br />
<br />
|tech= We electroporated pA13AM into ''DH5α'' and plated on ampicillin infused plates. We will know if the transformation was successful by the presence of red colonies as a result of ''mRFP'' in the plasmid. We found that our construction of pC13AY failed after running a digest screen as none of the lanes displayed bands at around 3kb, which corresponds to inserted ''PtrpC'' and ''T7 polymerase'' and 2kb corresponding to the vector. However, the gel may have been showing unique banding patterns for the construct, so another gel will be run to check for any supercoiling or linearized plasmids. We also submitted our constructs pC13BF and pC13BG for sequencing. Colony PCRs were done for pAK13AB, pAK13AC, and pAK13BB. A few pAK13AC and pAK13BB colonies were successful. <br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 5th<br />
|text= Eric Holmes and Arun Chakravorty presented for a group of scientific researchers (SILS)<br />
|author=Hannah<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 6th<br />
|text= After the previous failure of pC13AY construction, we recreated it with successfully screened parts. We redid a colony PCR for pAK13AB, which failed again. We will need to reconstruct pAK13AB. <br />
<br />
|tech= Yesterday’s transformation of pA13AM yielded no transformants, so we retried the transformation today. We ran a digest screen of ''PtrpC'' and ''T7 polymerase'' parts and found that the ''PtrpC'' used for our previous construction of pC13AY was of poor quality. We then set up another ligation of ''PtrpC'' and ''T7 polymerase'' using successfully screened parts. For fluorescence characterization, we double digested pAK13D and pC13P with SpeI and PstI for subsequent ligation with ''GFP'' and ''mRFP'' inserts. We then quantified our construct containing the <i>G. lucidum</i> homologous regions in pSB1C3, but we had a low yield. We will wait for a new miniprep kit to grow new cultures and perform the miniprep again. We redid a colony PCR for pAK13AB for extra colonies. They all failed so we need to reconstruct pAK13AB.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 7th<br />
|text= We received sequencing back for pC13BF and pC13BG and only the later was successful, so we restarted construction for pC13BF. We also ran a PCR to amplify ''GFP'' and ''mRFP'' inserts out of their respective plasmids.<br />
<br />
|tech= We designed primers for ''lox'' Gibson assembly and appended cut sites to genes flanked by ''lox''. We also found a possible viable kill switch in the Holin/Chitinase system. Our second try at transforming pA13AM was successful as shown by the presence of red colonies and we set up LB cultures for subsequent cloning. Sequencing showed that construction of pC13BG was successful and cultures were made for further use. However, sequencing of pC13BF was unsuccessful and we reran the PCR of pC13F in order to append BamHI and KpnI cut sites for ligation with pA13BE. We ran another digest screen for pC13AY but many lanes did not have bands so we will retry it tomorrow. We also amplified ''GFP'' from pC13Z and ''mRFP'' from pC13AK using primers 23 and 24 in a Q5 PCR. Afterwards, a gel screen on PCR products was run to verify that the correct genes were amplified. The gel image indicated consistent bands with lengths of 0.7 kb, thus the PCR to obtain ''GFP'' and ''mRFP'' inserts was successful! We made a rough draft of our protoplasting protocol to attempt in the near future, too. We then rummaged through previous notebook entries to figure out the problems with ''crtB'' and ''crtI''. <br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=August 7th<br />
|text=We helped [[Team:Ciencias-UNAM|Ciencias-UNAM]] get set to transfer their HTML website to the wiki. Hopefully we'll get up a tutorial for what to look out for on here soon!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 7th<br />
|text=The sensor has been connected! <br />
|author=Rebecca<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 8th<br />
|text= Cloning of the ''Cre'' gene and ''PpelA'' is going well, and we submitted ''PT7'' with ''GFP'' for sequencing, as well as pC13AS, pC13W, pAK13BB, and pAK13AC. <br />
<br />
|tech= We ran a PCR of pA13BW. The ''CRE'' gene amplified correctly and the sample was column purified, yielding 54.8ng/uL (with GC enhancer) and 49.9ng/uL (with non GC enhancer). We ran out of supplies to miniprep, so we pelleted pA13AM and pC13BG cultures and began to not so patiently await the arrival of a new miniprep kit. Our PCR of pC13F to append BamHI and KpnI cut sites yesterday failed completely, so we digested pC13F with KpnI to linearize it with an internal cut site in ''mRFP'' for another PCR to be run tomorrow. Another gel was run of the pC13AY digests and all lanes were blank. There may have been an issue in quantification by nanodrop that was reading over 1000 ng/μL that mislead us into using low volumes of DNA for digestions. We decided to electroporate with the remaining pC13AY to miniprep and redo gel screening. On a brighter note, our restriction cocktail digestion of pA13BE with BamHI, KpnI and SacI showed a band at 500bp, which is the approximate length of ''PpelA''. We also searched for a pAK13AN miniprep in the DNA storage box and for a glycerol stock of the same plasmid. We couldn’t find either. However, we did obtain a plate of colonies that had been transformed with pAK13AN. We picked colonies off this plate to miniprep and send off to sequencing. In addition, we column purified our inserts from the PCR run yesterday of pC13Z and pK13AL. We then quantified inserts after column purification. After, we digested backbones pAK13D and pC13P with SpeI and PstI and vector dephosphorylated both. The inserts, ''GFP'' in pC13Z and ''mRFP'' in pK13AL were then digested with XbaI and PstI. We ran a gel of backbones and inserts for confirmation of digests. Unfortunately, the results showed no bands from DNA. Perhaps there was not enough DNA from backbones for proper visualization. We also received materials for protoplasting today, and autoclaved or sterilized the supplies in whatever way we could. We made a filtration system such that all the filtering process during <i>G. lucidum</i> protoplasting will remain sterile. We made another stock of <i>G. lucidum</i> in CYM media, as well as 25 mL cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM. We made glycerol stocks of pC13M and pC13K from glycerol stocks. We submitted pAK13AC for sequencing and ran a Q5 PCR for amplifying insert for constructing pC13I from pC13A. <br />
|author=Jonlin <br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 9th<br />
|text= Cloning of the Cre gene is going well, and we may have found a useful limonene synthase gene! We are continuing the previously problematic cloning of ''GFP'' with T7 promoter-hopefully it works this time! Sequencing of pAK13AC unfortunately failed. <br />
<br />
|tech= We digested both the PCR products of ''Cre'' and pC13F with EcoRI and SpeI. We may have found a limonene synthase gene we can use. We ran another PCR to append cut sites to the linearized pC13F that ultimately failed with no bands visible on a gel. As our previous construction of pC13AY likely failed since no colonies being present on the new plate, we redid digestions of pC13E and pC13P to redo the construction. We also redigested both pAK13D and pC13P minipreps with SpeI and PstI since the gel from yesterday was inconclusive. We then ligated pAK13D with ''GFP'', pAK13D with ''mRFP'', pC13P with ''GFP'' and pC13P with ''mRFP''. We prepared pellets from our cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM cells which were stored in the fridge as we wait for our miniprep kit to arrive. Sequencing of pAK13AC has failed, and we will resubmit a miniprep of it. We plated stocks of pC13M and pC13K. <br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 10th<br />
|text= We began constructing pC13BH with Gibson assembly, as well as making our first attempt at protoplasting <i>G. lucidum</i>. Electroporations of fluorescence constructs was also done today. <br />
|tech= We did a fast ligation (30 minutes, room temperature) of ''Cre'' with pC13F and electroporated. The time constant was low, so the transformation may not have worked. We continued the ligation overnight. We were still waiting on a miniprep kit so we pelleted our pA13AM and pC13BG cultures to prevent further growth. We transformed with our new pC13AY ligation to hopefully get some colonies this time. We also ran a PCR of pC13F to append homologous regions to <i>G. lucidum</i> ''PgpdA'' for Gibson assembly to create pC13BH and another PCR to retry appending BamHI and KpnI cut sites to pC13F using half to reaction volume to see if efficiency would be improved. We also electroporated the 4 ligations for fluorescence from yesterday (pAK13D with ''GFP'', pAK13D with ''mRFP'', pC13P with ''GFP'' and pC13P with ''mRFP'') along with a control of pC13F to make sure that the electrocompetent cell stocks made yesterday are good. We plated the electroporated cells onto selective antibiotic resistance plates and incubated in 37 degree incubator overnight. We made our first attempt at protoplasting <i>G. lucidum</i> today. However we were having difficulty forming pellets of protoplasts, so we viewed the samples under a microscope. It appeared that the protoplasting attempt was unsuccessful. pC13M and pC13K plates were lawns on their plates. We set up overnight cultures of pC13AS, pC13W, and pC13Y, planning to miniprep and make glycerol stocks.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 10th<br />
|text= Five team members presented at the Ithaca Sustainability Center for citizens of Tompkins County.<br />
|author=Hannah<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 11th<br />
|text= We are starting cloning of limonene synthase and continuing cloning of the ''Cre'' gene. <br />
Cloning of fluorescent constructs is going well. <br />
|tech= The electroporation from the 10th did not work (there were no colonies). Electroporation was redone on the overnight ligation of ''Cre'' and pC13F and a better time constant was observed (even with poor cuvette drying). We transformed limonene synthase from 2013 kit plate 4. We discovered that we need spectinomycin to use the holin parts registry plates. We got colonies from the pC13AY transformation and cultured them for gel screening. For fluorescence, no colonies were found on pAK13AN and pAK13AO plates. We centrifuged potential pC13AQ cultures for 10 min at 5000g and poured off supernatant. We kept the cell pellets in the fridge for mini prepping tomorrow. We also found two colonies each on the PtrpC and ''GFP'' plate and PtrpC and ''mRFP'' plate. We picked off all the colonies and grew each up in 5 ml liquid LB with CM antibiotic. pC13M and pC13K plates are now ready for miniprepping. We could not miniprep the cultures of pC13AS, pC13W, and pC13Y due to running out of our miniprepping supplies.<br />
|author=Rafael<br />
}}<br />
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<div id="week9entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 9</h3><br />
<h5 class="centered">(08/12 - 08/18)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 12th<br />
|text= Cloning of PtrpC and ''GFP'' appears to be going well. Our initial attempt at protoplasting and transforming <i>G. lucidum</i> does not appear to have been successful.<br />
|tech= We still didn’t receive a miniprep kit so we pelleted the pC13AY cultures. The PCRs we ran on pC13F to append homologous regions and append cut sites may have been successful. DNA was present but in low concentrations that we could not check on a gel. We had transformants for pC13BX and pC13CJ so we ran a colony PCR and made cultures. The colony PCRs showed little to nothing on the gel, perhaps because Taq polymerase lost activity. We will miniprep the culture and use Q5. We also attempted a restriction cocktail digest today for both ''mRFP'' and ''GFP'' inserts. We cut pC13Z and pC13AK with Xba, PstI, SacI, and ApaLI. We also performed a colony PCR of pC13AT cultures and pC13AU cultures using primer 23 and 24 to verify length of inserts. The lengths should be around .7kb for each insert. When we ran a gel of the PCR products, the colony PCR showed a .7kb insert for pC13AT colony 1 and a .3kb insert for pC13AT colony 2. The bands for pC13AU were inconclusive. We will miniprep the potential pC13AT colony 1 and send for sequencing once the miniprep kit arrives. We digested our plasmid pAh13G with SphI in order to create linear DNA for fungal transformation. We attempted to transform this into what <i>G. lucidum</i> protoplasts we had generated, but the electroporation lead to arcing. Arching most likely resulted from the sodium citrate in the cell solution which we were instructed to use by the literature. Nevertheless, we plated the cells on hygromycin cym agar.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 13th<br />
|text= We continued cloning of ''T7'' with ''mRFP'', PtrpC with ''GFP'', and PtrpC with ''mRFP''. <br />
|tech= We spun down the pC13BX and pC13CJ cultures and put them in the fridge to be miniprepped. We also ligated ''T7'' with ''mRFP'', PtrpC with ''GFP'', and PtrpC with ''mRFP'' since restriction cocktail digests looked good from the gel yesterday. We then electroporated the ligations and plated them. The plates were left overnight in the incubator. We created another culture of <i>G. lucidum</i> this time in an effort to obtain solid pieces at the top of the media instead of mycelium floating throughout. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 14th<br />
|text= Homologous regions were successfully appended to ''PgpdA'', and cloning of the Cre recombinase appeared to be successful. We made another attempt at protoplasting, which was again unsuccessful. For characterization, we began cloning RBS into our constructs containing ''T7'', since the promoter need a ribosomal binding site to express in <i>E. coli</i>. <br />
|tech= We ran a PCR to append homologous regions to <i>G. lucidum</i> ''PgpdA'' for Gibson assembly with pC13F that succeeded as verified by a gel screen, but we accidentally used a RNA binding tube during purification and lost all the DNA. Needless to say, that mistake won’t happen again. Luckily, when we ran the same PCR again it was successful. We ran a colony PCR from the grown cultures of pC13BX and pC13CJ to visualize the insert using Q5. We got a positive insert for pC13BX. So this will be miniprepped and sent for sequencing once the miniprep kit is in. Unfortunately, no colonies appeared on the potential pAK13AO plate. We also decided to transform RBS (pA13CZ) off of a kit plate for subsequent miniprepping/digestion/ligation with ''GFP'' and ''mRFP''. The ''T7 promoter'' needs a ribosomal binding site in order to express in <i>E. coli</i>. We then retried the ligation of pAK13D with ''mRFP'' using a 6:1 insert to vector ratio. We attempted to protoplast a huge chunk of mycelium, but had difficulty forming a pellet again. At this point, we decided to enlist the help of Professor Gillian Turgeon, who performs fungal transformations in her lab. We made some hygromycin plates to test transformants, and digested pAh13G with Sph1 to linearize, but this time we column purified the product.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 15th<br />
|text= We ran a Gibson assembly to create pC13BH and transformed the construct.<br />
<br />
|tech= We ran a Gibson assembly of pC13F and <i>G. lucidum</i> ''PgpdA'' to create pC13BH and then transformed and plated the new construct. As our previous attempts to append BamHI and KpnI cut sites to pC13F failed, we again ran another PCR, but tried using four times the amount of dNTPs. We ran a PCR of pC13CJ once again from the cultures. The gel looks good for colony 4 and okay for colony 3. We will miniprep colony 4 and send it in for sequencing. We also made five 5ml liquid LB cultures of pA13CZ and miniprepped pC13AQ. We ran a digest screen of select pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BL, pC13F+pc13BM colonies with EcoRI and SpeI. Our initial gel didn’t show inserts (lengths of 130 and 230 base pairs) so we ran a second gel with 2% agarose. We still couldn’t see inserts, so we ran digest screens on all colonies (1-5) and ran a new gel. We saw very faint bands for some of them, and nothing for others. Additionally, Arun created a device to use the shaking power of our vortex to agitate a sample in the waterbath. We made LB cultures of pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13AC and pAK13BB. We set up new overnight cultures of pC13AS, pC13W, and pC13Y.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 15th<br />
|text=Rebecca looked more into the details of the pulse-width modulation and PID controller so that the heater can be turned on for certain periods of time for more even heating. <br />
|author=Rebecca<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 16th<br />
|text= The miniprep kit finally arrived so we were able to continue progress on our pC13BG, pC13AY and pC13AW constructs. To aid in our protoplasting, we decided to seek help from the Turgeon Lab.<br />
<br />
|tech= The miniprep kit arrived, so we finally were able to miniprep our pelleted pC13BG, pC13AY and pA13AM cultures. We got transformants for the Gibson assembly of pC13BH and we cultured them. The PCR to append cut sites to pC13F again failed, so we need to extensively troubleshoot this PCR. We made large cultures of pC13BX colony 1 and pC13CJ colony 4 to miniprep and sequence. We made electrocompetent stocks of BL21. The symbol is a purple star. We later met with Bradford Condon, a graduate student who works with Professor Turgeon. He gave us a few tips on our method, including using young mycelia, and avoiding the hydrophobic disc that forms on the top of our cultures for protoplasting. The advice was that we should blend <i>G. lucidum</i> which has been growing for an extended period of time, and make new cultures for a short period of time with the fungal "smoothie". We also miniprepped pC13BM, pC13BJ, pC13BL, and pC13BK 6 & 7. The LB stock of pAK13AC was made with the wrong antibiotic so we remade it. We also remade LB stock of pAK13AB. We miniprepped pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13BB, pC13AS, pC13W, and pC13Y. We plated pC13A, pC13D, pC13F, pC13K, pC13L.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 17th<br />
|text= PtrpC with ''GFP'' will be sent in for sequencing. We ran PCR of pAK13AC, pAK13BB, pAK13A (for both ''rbs'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but pAK13A both failed. We ran PCR of pAK13AC, pAK13BB, pAK13A (for both ''rbs'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but both pAK13A failed.<br />
<br />
|tech=Today, we made glycerol stocks of pC13AT and prepared them for sequencing. We then miniprepped pAK13AC. However, the miniprep qualities are poor so we will redo with a larger stock of cells. We ran a PCR of pAK13AC, pAK13BB, pAK13A (for both ''RBS'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but pAK13A failed. Thus, we retried PCR of pAK13A inserts from a fresh batch of minipreps. We prepared cultures of pC13S, pC13P, pC13Q, and pC13BG.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 18th<br />
|text= Cloning of ''T7 promoter'' with ''mRFP'' has been problematic - but we will keep trying. Minipreps of large stocks of pAK13AB, pAK13AC and pAK13BB yielded better results! <br />
<br />
|tech= Today we miniprepped pC13AT along with pA13CZ. We also ran a gel of PCR products of pAK13AO (5 colonies) in pSB1C3. Insert lengths should be around .7kb, but no bands showed up. We will rerun a gel using more DNA from the same PCR. We ran digest screens of pC13BM, pC13BJ, pC13BL, and pC13BK minipreps 6 & 7, which appear to have all worked. We miniprepped pAK13AB, pAK13AC, pAK13BB, pC13A and plasmid concentration was much higher this time around. We miniprepped pC13S, pC13P, pC13Q, and pC13BG, to prepare for cloning.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=August 18<br />
|text=Today, the dastardly trio of Arun, Hannah, and Eric were chosen to present at the regional iGEM competition!<br />
|tech=<br />
|author=Swati<br />
}}<br />
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<div id="week10entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 10</h3><br />
<h5 class="centered">(08/19 - 08/25)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 19th<br />
|text= We are beginning construction of ''T7 polymerase'' with an ''Anderson promoter'', and prepared PtrpC with ''mRFP'' and PtrpC with ''GFP'' for sequencing. Gel electrophoresis showed that pAK13AB, pAK13AC, pAK13BB and pC13A (both ''rbs'' + ''crtE'' and ''crtE'') are good! <br />
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|tech= We ran a PCR of the pC13BH minipreps to verify the success of the Gibson assembly on a gel. We also digested pC13AY in order to gel screen it and check if it is successful. We began constructing pA13AW by digesting and ligating pA13AM and pC13E. We also reran a gel of a colony PCR of pAK13AO. Next, we prepped pC13AT and pC13AQ for sequencing. We sent in pC13BJ, pC13BK, pC13BL, pC13BM minipreps 6 & 7, pC13AS, pC13W, and pC13Y for sequencing. We ran a gel for the PCR products from Aug. 17 and Aug. 18. Everything is good: pAK13AB, pAK13AC, pAK13BB, pC13A (both ''rbs'' + ''crtE'' and ''crtE''). We ran Q5 PCR of pC13N, pC13L, pC13S, pC13Q, and pC13W for creating inserts. We digested pC13S, pC13Q, and pC13W with X and P, and digested pC13BG, pC13D, pC13Y, and pC13P with S and P. We dephosphorylated and set up the ligations of those inserts and vectors to make pC13BQ, pC13BR, pC13CC, and pC13CD.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 20th<br />
|text= Cloning of PtrpC with ''T7 polymerase'' continues to be problematic. We are continuing cloning of ''PpelA'', last time’s ''T7 polymerase'' with an ''Anderson promoter'', and putting a strong RBS with ''GFP'' and ''mRFP''. We digested pC13I, pC13J, pC13N, pC13L, pAK13AB, pAK13AC, pC13F and pAK13D in preparation for ligating new products for the carotenoid pathways.<br />
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|tech= We transformed and plated the pA13AW ligation from yesterday. We also retried the PCR to append KpnI and BamHI cut sites to pC13F again, using the newly received Q5 mastermix and higher annealing temperatures. We ran a massive gel screen and found that the cut site PCR and Gibson assembly may have been successful, but that the construction pC13AY most likely failed. We then digested pC13F with cut sites appended with BamHI and KpnI for ligation with pC13BE to make pC13BF. We digested pA13CZ with SpeI and PstI, which is to be ligated with ''GFP'' from pC13Z and ''mRFP'' from pC13AK. We ligated the Xpa1, Pst1, Spe1, Apa digested pC13AK and Spe1 and Pst1 digested and dephosphorylated pC13BG to make pC13CG, which we transformed into BL21-A1 cells to check transformation efficiency. We also tranformed into DH5α and plated both for comparison. The sequencing results of pC13BJ 7, pC13BK 6, pC13BL 7, and pC13BM 6 confirmed that we were successful, so we made new cultures to create glycerol stocks from, but the initial cultures were thrown out and the plates were difficult to re-pick from, so we hope that cells made it into the culture and have the correct plasmid. We did a PCR clean-up for pAK13AB, pAK13AC, pC13A, pC13A, pC13N, pC13L. We digested pC13I, pC13J, pC13N, pC13L with XbaI & PstI, pAK13AB & pAK13AC with EcoRI & PstI. We also digested pC13F with XbaI and PstI, also with EcoRI and PstI, as well as pAK13D with XbaI and PstI. We transformed ligations of pC13BQ, pC13BR, pC13CC, and pC13CD into <i>E. coli</i>. We also interpreted sequencing results of pC13AS, pC13W, and pC13Y - the latter two look good, and pC13AS may have an overlap, suggesting a mixed population of plasmid.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 21st<br />
|text= Weird happenings in lab: what we thought was PtrpC with ''GFP'' turns out to be PtrpC with ''mRFP''! Good news: ''PgpdA'' and ''pC13F'' with homologous regions for Gibson are good! We tested the transformation efficiency of our new T7-ready bacteria, BL21-A1 as well. We ligated and electroporated pC13AF, pC13AG, pAK13CR, pAK13CS from other digestions, and plated them overnight. <br />
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|tech= We made cultures of the successfully transformed pA13AW and ran a gel screen of the digests of pC13F and pC13BE and found that they did not have enough DNA to be visible. The <i>G. lucidum</i> ''PgpdA'' and pC13F parts with homologous regions needed for Gibson assembly and found them successful. We then ligated and transformed pC13BF although it sparked during electroporation, so we will most likely have to retransform. A sequence confirmation came back for pC13AQ! pC13AT turned out to be PtrpC and ''mRFP''.Thus, we redigested PtrpC in pC13P and pCg13Y with SpeI and Pst1-HF. pCg13Y will be used to create pC13CB. We also redigested pC13Z, pC13AK, and pC13AQ with Xba, Pst1-HF, ApaI and Sac1-HF in order to get necessary inserts. We threw away the cultures of ''pC13BJ" 7, ''pC13BK" 6, ''pC13BL" 7, and ''pC13BM" 6 from yesterday and didn’t make glycerol stocks because we were not sure if we got the correct colonies. We will transform the sequence confirmed minipreps when more DNA is needed. We attempted site directed mutagenesis of ''ptrpC'' + ''hph'' + ''ttrpC'' in pAh13G. The extension time was a little short for pAh13G so it might not work. We ran gel a gel, which had a band at 100 base pairs, meaning it didn’t work. We will attempt this again tomorrow with a longer extension time. Colonies of BL21-A1 and DH5α with pC13CG grew. BL21-A1 seems to have a higher transformation efficiency.We started cultures and performed a colony PCR from the DH5α plate. We ran a gel of the colony PCR and pC13CG 1, 3, and 4 had the correct band at about 1.8 kb. pC13A (''rbs'' + ''crtE'') and pC13F (XbaI,PstI) did not have enough DNA. As a result, ligations for creating pC13I and pC13J are on hold. We ligated and electroporated pC13AF, pC13AG, pAK13CR, pAK13CS from other digestions, and plated them overnight. We found the the transformations of pC13BR and pC13BQ were too efficient and had to restreak the plates in order to isolate single colonies. We ran colony PCRs of pC13BU, pC13CC, and pC13CD, and at least two copies of each construct seem to be successful!<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 22nd<br />
|text= We did a bunch of ligations and are continuing cloning of ''PgpdA'', ''Anderson promoter'' with ''T7 Polymerase'', ''GFP'', geneticin resistance, and a strong RBS. We ligated pC13I and pC13AF. <br />
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|tech= We redid the Gibson assembly of pC13BH trying 1:2 and 1:6 vector to insert ratios and transformed it along with pC13BF as it sparked when we tried yesterday. We also miniprepped our pA13AW cultures. After, we vector dephosphorylated pA13CZ, pC13P, and pCg13Y for fluorescent characterization constructs. We then ligated ''PgpdA'' with ''GFP'', pC13AQ with pC13Y, pC13P and ''mRFP'', pC13P with ''GFP'', pA13CZ with ''GFP'', and pA13CZ with ''mRFP''. All ligations were then electroporated and incubated overnight. We sent pC13BX and pC13CJ to sequencing. We digested and ligated pC13CJ and pAK13D and electroporated into DH5a cells. We miniprepped and made glycerol stocks of pC13CG 1,3,4 (labeled TEMP). We will make sure to go back and throw out extras and failed attempts after sequencing results return. We sent pC13CG 3 in for sequencing, using the last of our DNA, meaning if we need more it will have to come from the glycerol stock. We performed our second attempt at site directed mutagenesis of pAh13G with a longer extension time. We recreated and digested inserts pC13A (''rbs'' + ''crtE'') and pC13F (XbaI, PstI). We ligated pC13I and pC13J. pC13AF electroporation did not yield any colonies. We made glycerol stocks of pC13AG, pAK13CR, pAK13CS, pAK13BU, pC13CC, and pC13CD and put them at -80 degrees C. We miniprepped the successful pAK13BU, pC13CC, and pC13CD and prepared them for sequencing. We also ran colony PCRs of pC13BR and pC13BQ, and set up overnight cultures of the successful constructs.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 23rd<br />
|text= Cloning of RBS + ''GFP'', RBS + ''mRFP'', ''PT7'' + ''GFP'' + PtrpC + geneticin resistance, PtrpC + ''mRFP'', and PtrpC + ''GFP'' appear to be going well. <br />
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|tech= Today we made liquid cultures of and ran colony PCRs of pA13DC, pA13DB, pC13AU, pC13AT, and pC13CB. pC13CF did not grow any colonies. We then ran a gel of the PCR products. pC13CB colony 5, pC13AT colonies 1-3 and 5, pC13DC colonies 2-5, pC13DB colonies 1-5, and pC13AU colonies 1 and 5 are looked like they had the proper insert lengths from the gel image. Sequencing results came back confirming that pC13CG 3 worked We digested our site directed mutagenesis attempt from yesterday with DpnI and stored it in the fridge. We ran a gel combined with other DNA from other team members, which resulted in a smeared band that was in the correct place. We believe this will be successful, and we will transform our site directed mutagenesis product as soon as desalting membranes arrive. We mini-prepped and plated pC13AG, pAK13CR, pAK13CS. We miniprepped pC13BQ and pC13BR, and submitted pC13CC, pC13CD, pC13BQ, and pC13BR for sequencing. We ran PCRs of the inserts in pAK13BB and pAK13BU to prepare for further cloning.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 24th<br />
|text= Running into some problems with ''PgpdA'' cloning. PCR confirmed that pC13AG and pAK13CR worked! We picked more colonies of pAK13CS for screening. <br />
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|tech= We cultured transformants for our Gibson assembly of pC13BH using 1:2 vector to insert ratios, but got no colonies for the 1:6 ratio Gibson and the pC13BF ligation. We also digested our pA13AW minipreps for gel screening. Our PCR confirmed that pC13AG and pAK13CR worked. We picked more colonies of pAK13CS from electroporation plate to screen more colonies. <br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 25th<br />
|text=The extra colonies of pAK13CS from yesterday failed. We will have to restart pAK13CS. We are also recreating inserts from pAK13AB to make pC13AF. <br />
|tech=The extra colonies for pAK13CS that we made yesterday all failed. We will need to restart pAK13CS. We are recreating insert from pAK13AB for recreating pC13AF. <br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 25th<br />
|text=Christine's back! She started looking at the components for a humidity sensor and placed an order for a mistmaker which would be able to control humidity levels. In the meantime, Sara studied the details of the heating circuit which utilizes a transistor to amplify signals and switches it such that the resistor could be used as the heating element. <br />
|author=Rebecca<br />
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<div id="week11entry" class="nb-week"><br />
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<h3 class="centered">Week 11</h3><br />
<h5 class="centered">(08/26 - 09/01)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 26th<br />
|text=Cloning of pA13AW seems to be successful and will test to see if pC13BH also worked. We ran a PCR and digested pC13L to prepare insert for recreating pAK13CS. We recreated ligation of pC13AF.<br />
|tech= The transformed colonies of pC13BH had the ''PgpdA'' insert amplified via colony PCR and gel screened to check for the correct length. The gel however was blurry, so the cultures were miniprepped for digest screening. Gel screen pA13AW digested with XbaI and PstI. Gel results indicate that the cloning was successful! We then amplified the ''T7 polymerase'' gene for more insert DNA. We ran a PCR and digested pC13L to prepare insert for recreating pAK13CS. We recreated ligation of pC13AF. We looked at sequencing results for pC13BQ, pC13BR, pC13CC, and pC13CD - all looked good, other than some minor differences between our copies of the trpC promoter and terminators and the sequences in the online database.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 26th<br />
|text=Our materials are here and everyone in the fabrication group has returned to Ithaca! This week, Manny and Mac focused their efforts on getting the plastic cut to the proper dimensions. Unfortunately, this proved more to be more difficult than anyone could have imagined. After transporting a rather hefty 48"x48" piece of plastic across campus from Weill Hall to the Emerson Lab machine shop, we were told that we would have to take it elsewhere to have it cut (plastic is bad for blades). Disappointed, but never defeated, we decided to try our luck with another machine shop. Also, carrying plastic around campus isn't much fun, so we held it on the roof of a car instead (not recommended). Fortunately, no traffic violations were received, and the Clark hall machine shop was more than happy to cut up our plastic for us. <br />
|author=Mac <br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 27th<br />
|text=Digest screens indicate that cloning pC13F and the ''T7 polymerase'' PCR worked. We are working on the ''cre'' and ''lox'' constructs. pC13AU, pC13AT, pC13CB, pA13DC, pC13AG, and pA13DB were miniprepped and prepared for sequencing. We prepared insert from pAK13CR for creating pC13CV. <br />
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|tech= We made cultures of pC13BX colony 1 and pC13CJ colony 4 to make glycerol stocks. We ran PCR of ''lox'' sites to amplify by Gibson. We ligated pAK13D with limonene. We preppred ''cre'' for reverse sequencing. We transformed ''holin'' off of kit plate and plated it. Digested pC13BH preps using EcoRI and PstI and screened alongside ''T7 polymerase''. Results indicate that only the ''T7'' PCR worked. The backbone pC13F and pA13AM were digested with XbaI and PstI, retrieving the ''T7 polymerase'' gene from the latter. Gel results for the digestions revealed correct banding patterns only for pC13F. We also miniprepped and prepared for sequencing pC13AU, pC13AT, pC13CB, pA13DC, and pA13DB. We prepared insert from pAK13CR for creating pC13CV. We submitted pC13AG for sequencing. We ran PCRs of pAK13BB and pAK13BU, and digested the inserts with EcoRI and PstI. We also digested pC13CD with SpeI and PstI.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 28th<br />
|text=Sequenced pC13BH and ligated ''T7 polymerase'' into a variety of vectors. ''lox'' 2 worked with Gibson, but ''lox'' 1 did not. We created large batch of vectors pC13F and pC13D. The sequence of pC13AG is confirmed! <br />
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|tech= One colony of pC13BH had a good banding pattern and was submitted for sequencing. Digested ''T7 polymerase'' PCR with XbaI and PstI and pC13F with BamHI and KpnI (sites were appended via PCR). pC13F that was digested with XbaI and PstI as well as with BamHI and KpnI were dephosphorylated for ligation. After confirming ''T7 polymerase'' PCR on a gel, it was ligated into pA13AM and pC13P. Additionally, ''Pc + T7 polymerase'' was ligated into pC13F and ''PpelA'' was ligated into pC13F. We ran those PCR's for the Gibson of pC13BN on a gel. ''lox'' 2 worked, but ''lox'' 1 did not, so I purified ''lox'' 2 and reran the PCR of ''lox'' 1 (it is in the small fridge labeled ''lox'' 1). I also made glycerol stocks of pC13BX and pC13CJ so that we have those in case we need to do more minipreps on them. We also had transformants for pC13CN so I made cultures with spectinomycin for all of them and ran a colony PCR on them. The colony PCR did not work due to incorrect protocol. We do not have the correct primers to do a PCR on this plasmid because its in Bbb format, so we just have to assume the plasmid is present because the cultures are growing in Spec. Tomorrow we need to miniprep those cultures that are in the incubator. We blended <i>G. lucidum</i> and made two 100mL cultures with 75mL cells and 25 mL cells. We will now let these grow for 2 days before attempting protoplasting once more. We created large batches of vectors (pC13F (XbaI,PstI) and (EcoRI-HF,PstI), pC13D (XbaI,PstI)). pC13AG sequence is sequence confirmed! We tried to dephosphorylate, but mistakenly used DNA Ligase on pC13F and pC13Q instead, so we’ll have to redo that tomorrow.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 29th<br />
|text=Transformed our ''T7 polymerase'' ligations. We ligated pAK13CS and pC13CV. We electroporated and plated pC13I, pC13J, pAK13CS and pC13CV -- pC13J unfortunately arced…<br />
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|tech=Today we electroporated ''Pc + T7 polymerase'' in pC13F, ''PpelA'' in pC13F, and ''T7 polymerase'' in pA13AM and pC13P. We prepared pAK13AB and pC13AG for glycerol stock. We ligated pAK13CS and pC13CV. We electroporated and plated pC13I, pC13J, pAK13CS, pC13CV. pC13J arced, so we ran a new Q5 PCR to begin preparation of insert from pC13A. We re-digested pC13Q with XbaI and PstI and pC13F with EcoRI and PstI, and subsequently dephosphorylated pC13CD and pC13F. We set up ligations to create pC13BC, pC13BV, and pC13CE.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 30th<br />
|text=Cultures from yesterday’s ''T7 polymerase'' ligation transformations were made. We transformed the ''lox'' Gibson products. pC13AU and pC13AT were successfully sequenced today. We did a PCR with Phusion polymerase in an attempt to amplify the insert in pC13J. <br />
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|tech= Set up cultures for ''Pc + T7 polymerase'' in pC13F, ''PpelA'' in pC13F, and ''T7 polymerase'' in pA13AM and pC13P and ran a colony PCR for each. Each PCR was visualized on a gel; only ''PpelA'' in pC13F appeared to work. We quantified DNA: ''lox'' 1: 72ng/uL, ''lox'' 2: 34ng/uL, pC13CN 640.9 & 820.9ng/uL. We ran Gibson on the ''lox'' PCRs, transformed them into DH5a cells. We made glycerol stocks of pC13CN. We have pC13CK transformants, ran colony PCR and made cultures. We got reverse sequencing on cre and it looks good. We inoculated a culture of DH5a for electrocompetent stocks. pC13AU and pC13AT was sequence confirmed today. However, for pC13CB, PtrpC and ''nptII'' BB re-ligated back to itself pA13DB colony 1 and pA13DB colony 4, pA13DC colony 3 and pA13DC colony 4 all failed to sequence. We discarded all of our pC13CG glycerol stocks except for the successful pC13CG 3, and met with members of Professor Turgeon's lab. We also blended our 25 mL culture from 2 days prior, and made new 100ml cultures with (75 mL and 25 mL, again.) We prepared pAK13AB and pC13AG for glycerol stock. We made glycerol stock of pAK13AB and pC13AG. We redid a PCR with Phusion polymerase in an attempt to amplify ''rbs'' + ''crtE'' (pC13J). We then made overnight E. Coli cultures of pC13I, pC13AF and pC13CV. We transformed the ligations of pC13BC, pC13BV, and pC13CE into <i>E. coli</i>, but both attempts for pC13CE arced, so we started another ligation for pC13CE.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 31st<br />
|text= We are continuing cloning of the carotenoid genes and ''lox'' sites. The pC13BN construct looks good. We double digested pC13Z and pC13AK with Xba, PstI, Sac, and ApaLI since we were running low on insert DNA for ''GFP'' and ''mRFP''. We began a new attempt at protoplasting, now with the help of members of the Turgeon Lab. We digested the following miniprep products: pC13I, pC13AF and pC13CV. We also electroporated pAK13CS. The PCR of pC13J from yesterday is good! <br />
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|tech= We ran colony PCR of pAK13CK on gel and nothing appeared. This could be because we used a janky Vent protocol. We miniprepped liquid cultures of pC13CK. A digest screen was run on the minipreps, but they did not look good. We will redo cloning for the construct. In other news, we got pC13BN transformants! We ran a colony PCR and grew up liquid cultures of pC13BN. The colony PCR looks good, and we will miniprep liquid cultures of pC13BN tomorrow. We made electrocompetent DH5a stocks, which are marked with blue circles with a line through it. Lastly, we double digested pC13Z and pC13AK with Xba, PstI, Sac, and ApaLI since we were running low on insert DNA. The gel for we ran for the double digestion looked good since we got 4 distinct bands for ''GFP'' and ''mRFP'' each. Next, we column purified pC13Z and pC13AK double digests and stored them in the freezer for later use. Today we attempted the first day of protoplasting and transforming <i>G. lucidum</i> in the Turgeon Lab. We used both our 3 day old and 1 day old cultures of <i>G. lucidum</i> in this attempt. Our initial pellet of both cultures appeared about the same size, though the younger one may have been slightly larger. We observed that the cells didn’t look like protoplasts, as they weren’t round and were smaller/very few in number. We looked at the solution after filtering and were able to see protoplasts, so it appears that something didn’t work with buffer/centrifuging. We miniprepped and made glycerol stock of the overnight cultures from yesterday--pC13I, pC13AF and pC13CV. We then digested these products. We electroporated for pAK13CS and plated the culture on Ampicillin plates. In addition, the PCR of pC13J worked! The transformations for pC13BC and pC13BV were too efficient, so we had to restreak them out to single colonies.<br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 1st<br />
|text= We are putting ''PgpdA'' with geneticin resistance, PtrpC with geneticin resistance, and PtrpC with hygromycin resistance between the ''lox'' sites. We miniprepped pC13BN. A Vent colony PCR of pC13CB colonies 1-5 was run to confirm successful cloning. We finished our attempt at protoplasting, once again unsuccessful, likely due to contamination on the fungal cultures. We ran a digest screen for pC13I, pC13AF and pC13CV, but only pC13AF (colony #2) appeared to work. We then ran a PCR with Vent for pC13I, pC13AF, pC13CV and pC13L, but they all failed. We transformed the new ligation for ''pC13CE'' into <i>E. coli</i>.<br />
|tech= We ran a PCR for amplification of pC13Y, pC13BQ, pC13BR, and pC13CD to put between the ''lox'' sites. The PCR products were then cleaned and quantified. We redid the ligation of pC13D with pC13CJ to form pC13CK. We electroporated the ligation into DH5a. We miniprepped the pC13BN 2 cultures. We then ran a Vent colony PCR of pC13CB colonies 1-5. We also grew up 5ml cultures of pC13CB colonies 1-5 and left them in the incubator overnight. We finished our attempt at protoplasting <i>G. lucidum</i> with the help of members of the Turgeon lab, with hygromycin as our selection marker, and we left the plates in the Turgeon lab incubator. We then combined our three blended cultures and blended again to make a new culture for tomorrow. We planned out a protoplasting schedule for this week to try running the protocol under different conditions since it takes a long time to get results. We believe there might be bacteria growing in the fungal cultures we are using for protoplasting since phosphinothricin does nothing to bacteria and we could not blend the fungi under sterile conditions. We will put ampicillin in later cultures to kill bacteria, which shouldn’t affect <i>G. lucidum</i> because only bacteria will have peptidoglycan cell walls. We did a PCR clean up for pC13A. We ran a digest screen for pC13I, pC13AF and pC13CV, but only pC13AF (colony #2) appeared to work. We then ran a PCR with Vent for pC13I, pC13AF, pC13CV and pC13L, but they all failed… <br />
|author=Rafael<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 1st<br />
|text=Lydia's back too! Discussed what we have been doing for the past few weeks and immediate goals. <br />
|author=Rebecca<br />
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<div id="week12entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 12</h3><br />
<h5 class="centered">(09/02 - 09/08)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 2nd<br />
|text=Miniprepped ''PtrpC'' + ''T7 polymerase'', the ''PpelA'', the ''Anderson promoter'', and the ''Anderson promoter'' + ''T7 polymerase''. We are preparing to send pC13BN to sequencing. A Vent PCR was done on pC13CB to verify successful cloning. We began a new transformation attempt, now with fresh solutions to work with. However, this also seemed unsuccessful. We also ran a Vent PCR with the addition of MgSO4 for pC13AF, pC13CV, pC13I and pC13L minipreps--they all failed. Another Vent PCR was run to check inserts in pC13BC, pC13BV, and pC13CE, but resulted in no bands.<br />
|tech= We got colonies for pAK13CK and ran a colony PCR. The PCR didn’t show anything, but the primers are not ideal, so we will miniprep and digest screen. We digested PCRs of pC13BR, pC13BQ, pC13CD, and pC13Y with KpnI, HindIII. They were all cleaned. We digested miniprep of pC13BN with KpnI and HindIII. This was cleaned and dephosphorylated. We prepped pC13BN miniprep for sequencing with primer 31. Miniprepped pC13AY, pC13BF, pA13AM, and pA13AW cultures and will quantify tomorrow. We prepared pA13DC colonies 3 and 4 and pA13DB colonies 1 and 4 for sequencing Tuesday morning. We then reran a Vent PCR for pC13CB and grew up liquid cultures of the same colonies. We ran a gel for pC13A which showed that the PCR failed. We then redid a Vent PCR with the addition of MgSO4 for pC13AF, pC13CV, pC13I and minipreps of pC13L. We made 5 pAh13G cultures from glycerol stocks, and we electroporated pC13BJ, pC13BK, pC13BL, and pC13BM to make glycerol stocks. All transformations except pC13BM arced, but we are going to plate them anyway. We also attempted protoplasting again, using fresh enzyme osmoticum and STC. We couldn't really see mycelium at the beginning, and we could not see cells when we attempted to count them, possibly due to our mycelium being too old or having depleted its media. We made a new culture with a blended cell mass and ampicillin. We also made more CYM plates and media, and prepared a plan to test the sensitivity of <i>G. lucidum</i> to antibiotics and antifungals.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 3rd<br />
|text= Digest screen of ''PtrpC'' + ''T7 polymerase'' and the ''Anderson promoter'' + ''T7 polymerase'' showed good expected banding pattern. The pC13CK construct is correct. A gel of pC13CB PCR products was run and was inconclusive. We repeated the PCR from yesterday for pC13AF, pC13CV, pC13I and pC13L but using the Q5 polymerase. Except for pC13AF from colony #2, all else failed. We attempted to protoplast again, this time with emphasis on determining what our issue is in hopes that we can fix it.<br />
|tech= We quantified digestions and ligated pC13BQ + pC13BN, pC13Y + pC13BN, pC13BR + pC13BN, and pC13CD + pC13BN. We electroporated the ligations. We miniprepped pC13CK cultures and ran digest screen, which was successful. The construct is correct. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin'', which was unsuccessful. The pC13AY, pA13AM, and pA13AW preps were digested with EcoRI and PstI and run on a gel. Results indicate that pC13AY and some of the pA13AW colonies worked. The gel of Vent PCR of pC13CB did not turn out too well. We got a bunch of lanes with smeared rather than distinct bands. We then ligated and transformed ''T7 promoter'' with ''mRFP'' to obtain pAK13AO and ''PgpdA'' with ''GFP'' to create pC13CF. We ran a Q5 PCR and gel for pC13AF, pC13CV, pC13I and pC13L. Sadly, they all failed except pC13AF from colony #2. We are concerned with what’s going on. The PCR for pC13A failed again too. We miniprepped our pAh13G cultures. Our new fungal culture doesn’t have nearly as much mass in it, leading us to believe that the old cultures were almost definitely contaminated with bacteria. Our transformed pC13BK and pC13BM grew into masses covering the plates rather than colonies, and the others didn’t grow. We restreaked pC13BK and pC13BM hoping for single colonies. We attempted a diagnostic protoplasting, determining that pellets formed are likely from contaminations, as the new cultures with ampicillin did not produce pellets. We also tried to manually extract mycelium and enzyme digest it, but microscope visualization did not show signs of protoplasting having occurred. Even after filtering through one layer of cheesecloth and nylon, there was no appearance of protoplasts.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 3rd<br />
|text=Christine, Rebecca and Sara met up with Dr. Land again and we learnt more about setting up PID controls. Went to the lab next where Christine and Sara set up the heating element circuit, Rebecca soldered pins onto the arduino and Lydia drew up the circuit design.<br />
|author=Rebecca<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 4th<br />
|text= Transformed the ''PpelA'' and ''Anderson promoter'' + ''T7 polymerase'' into pSB1C3 as well as digest and ligate other promoters. The pC13BN construct was incorrect, so we will have to redo all of the Gibson transformations for the ''lox'' constructs. On a brighter note, sequence confirmation of pA13DB and pA13DC came back today!<br />
<br />
|tech= We got transformants for all ''lox'' constructs. We got sequencing back for pC13BN and it was incorrect, so we will redo the Gibson transformation and all the subsequent ''lox'' constructs. We also electroporated pC13BF and pC13AZ and digested pC13F and pA13AW with XbaI and PstI for ligation to make pC13AZ. pC13F and pC13BE KpnI and BamHI digests were then ligated to make pC13BH. pA13DB colony 1 and pA13DC colony 4 were sequence confirmed today. Lastly, we made cultures from restreaked pC13BM and pC13BK plates.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 5th<br />
|text= Today in lab we gel screened a bunch of old minipreps to see if they were still viable and made cultures of the ''Anderson promoter'' + ''T7 polymerase'' in pSB1C3. We digested the ''lox'' site constructs in order to ligate with pC13BN. In a meeting with Professor Turgeon we decided that we would leave <i>G. lucidum</i> behind for now and work with <i>Cochliobolus</i>, which her lab transforms regularly and has more expertise with.<br />
<br />
|tech= The Gibson was digested with DpnI and electroporated into DH5a. We are growing DH5a for electrocompetent stocks. We did a PCR of pSB1C3 to append cut sites in order to ligate with ''holin''. We digested pC13Y, pC13BQ, pC13BR, and pC13CN for ligation with pC13BN and made cultures of three pC13AZ colonies. Gel screen of pC13F and pA13AW appeared successful and were then column purified. We also gel screened pC13P miniprep, SpeI and PstI digested pC13P, PCR of ''PgpdA'' for homologous regions, PCR of pC13F for homologous regions, and BamHI/KpnI digested pA13BE to verify the viability of old minipreps. We then amplified ''T7 polymerase'' from pC13E. We did a Q5 PCR for pC13CV, pC13I and pC13L. Our plates from transformation on Saturday showed conidia growth on the controls with no hygromycin. None of the plates with hygromycin showed any growth. We decided that we will now use <i>Cochliobolus</i> to protoplast and transform after meeting with Professor Turgeon again. To determine if the DNA is in the fungal genome and stable, we will plate on non-antibiotic and antibiotic plates, then replate transformants from the antibiotic plate onto non-antibiotic plates and back onto antibiotic plates. Since protoplasting is failing, we will turn to agrobacterium for <i>G. lucidum</i> transformation in the future. We will also check for homology between the promoter gpd in <i>G. lucidum</i> and <i>Cochliobolus</i> to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after <i>Cochliobolus</i> gpd by cutting into that region in the genomic promoter to transform. We also created glycerol stocks of pC13BM and pC13BK cultures and miniprepped them to submit for sequencing. We set up new cultures of colonies from transformation of pC13BC, pC13BV, and pC13CE, and ran colony PCRs of them (using our brand new tube of Q5 polymerase!) to check for inserts.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 6th<br />
|text= Most of old minipreps were no good and the ''PpelA'' transformation failed. The ''Anderson promoter+T7 polymerase'' gel results looked good and were submitted for sequencing. Oh no, the PCR of pC13CV, pC13I and pC13L failed again--we suspect something is wrong with our reagents. Use the new dNTPs from now on!<br />
<br />
|tech= We digested, ligated, and transformed to create pC13DD. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin''. Gel results indicate that pC13P miniprep, pC13P SpeI/PstI digests, ''PgpdA'' PCR for Gibson and the pA13BE BamHI/KpnI/SacI digest are not viable, but the pC13F PCR for Gibson might be okay. pC13BF transformation failed as well. The pC13AZ cultures were digest screened; results were good, so they were sent for sequencing. pC13P was plated and pC13E was cultured. We submitted pC13AF (colony #2) for sequencing. The Q5 PCR from yesterday--pC13CV, pC13I and pC13L--was a failure. We suspected that something is wrong with our reagents. Use the new dNTPs from now on! We ran PCRs to prepare the necessary parts for our Gibson assembly to produce parts with flanking <i>G. lucidum</i> homologous regions. We spun down the cultures of pC13BC, pC13BV, and pC13CE and stored them in the fridge.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type= anim<br />
|head=September 6th <br />
|text= We met at Flora Rose house to decide on an outline of the presentation and also decide a timeline.We met for the first time to discuss a preliminary outline for the presentation. Hannah will be handling all the human practices elements, Eric will be explaining relevant biological background and strategies, and Arun will be presenting all of our characterization data (hopefully!). We made a detailed structure which will include an introduction about styrofoam pollution and ecovative. Then we will move on to a brief overview of our wetlab work. It was clear that the wetlab portion should be divided into 3 main components. The components would be fungal toolkit, carotenoid pathway, and antifungals. Finally, we will end with results, human practices, and acknowledgments to all our beautiful advisors and sponsors.<br />
|tech= <br />
|author=Arun<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 7th<br />
|text= Digested and ligated the ''Anderson promoter+T7 polymerase'' into pSB1C3 and pSB2K3 as well as start cultures from glycerol stocks and transformation and gel screened the ''Anderson promoter'' + ''T7 polymerase''. We transformed ''afp1'' and ''Cht1_2''. For fluorescence, we electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ to obtain more plasmid DNA. We redid a Q5 PCR for pC13CV, pC13I, pC13AF and pC13L with the new dNTPs--PCR products were all bad except for pC13L.<br />
<br />
|tech= We did Q5 PCR to amplify pSB1C3 for Gibson with ''afp1'' and ''Cht1_2''. 3uL of PCR product was run on the gel, found to correct, and was cleaned. We ran a Gibson of pSB1C3 with both pC13CL and pC13BA. Both of these were transformed, ''Cht1_2'' into DH5a (time constant 4.82) and ''afp1'' into BL21 (time constant 4.84). We made glycerol stock of pC13BN. We ran a gel of pSB1C3 PCR from yesterday, which was successful, and performed a PCR clean up. We digested pSB1C3 and pC13CN with HindIII and BglII. We ligated these together to create pC13CO and transformed it into DH5a (time constant 4.82). We miniprepped pC13BN culture and did a double digest on pC13BN culture with KpnI and HindIII to see if internal cut sites were present. There was some digestion that occurred when it was run on a gel, so we are confident the sequence is correct. We double digested pC13BN again with KpnI and HindIII. We ligated it with pC13Y (time constant 4.86), pC13BR (4.86), pC13BQ (4.90), pC13CD (4.80). We got transformants for pC13DD. A colony PCR was run and pC13DD colony 3 looked good. This colony was grown in liquid culture for miniprepping and sequencing. We grew cultures of pC13BX, pC13CK, and pC13BN for submission (pC13BX submitted directly; pC13CK and pC13BN need sequence confirmation first). pC13BF transformants were cultured, pC13AZ cultures were digested with EcoRI/PstI and gel screened; results were good, so they were submitted for sequencing. Digested pK13AL and pA13AW with XbaI/PstI for ligation to make pK13AX. pC13P and pC13E were digested with EcoRI/PstI and ligated to make pC13AY. Cultures of pC13Q and pC13BE were made from glycerol stocks. We electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ. We also ran a Q5 PCR of pC13CB and pAK13AO using standard BioBrick primers 23 and 24 in order to confirm lengths of inserts. We redid the Q5 PCR with the new dNTPs for pC13CV, pC13I, pC13AF and pC13L--pC13CV, pC13I and pC13AF were bad, but pC13L was good! We suspect that electroporation did not go well to begin with. We reran the 2-step Q5 PCR for pC13A. We ran a gel of our PCR to create Gibson assembly parts, and it appeared unsuccessful. We ran the colony PCRs of pC13BC, pC13BV, and pC13CE on a gel, and only observed bands for a few pC13BC colonies.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=September 7th<br />
|text= Five team members went to the Ithaca Sciencenter and taught a group of 18 children and 17 parents about the power of DNA.<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type= anim<br />
|head=September 7th <br />
|text= After researching we found out that the powerpoint presentation should be centered around design and images rather than content. We decided to meet tomorrow to discuss possible design layouts and schemes. We looked at many examples of presentations and considering our project, we think we should have a very modern and green theme. Arun will explore with this later on.<br />
|tech= <br />
|author=Arun<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 8th<br />
|text=We met super early in the morning (10 am!) for brunch and some delicious cake. We also got work done and made an entire storyboard for the presentation. Our golden (or perhaps orange) idea of the day was a growing carrot representation for the carotenoid pathway. <br />
|tech=<br />
|author=Eric, Arun <br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 8th<br />
|text= We miniprepped ''PpelA'', PtrpC, and ''TtrpC''. We got transformants with ''afp1'' and ''Cht1_2'' and for all of the ''lox'' site constructs. However, only the pC13CO looked good based on a gel screen. We will sequence the ''lox'' construct. We also miniprepped pC13CV, pC13I, pC13AH and pC13AG. <br />
<br />
|tech= We got transformants from the ''afp1'' and ''Cht1_2'' Gibsons. There were a lot of red colonies and very few white ones, and an attempted pick and streak of the white colonies was performed. We got transformants for pC13CO, pC13BN + pC13CD, pC13BN + pC13Y, pC13BN + pC13BQ, and pC13BN + pC13BR. A colony PCR was performed, and a gel of the PCR products looked good for only pC13CO colony 2. No positive inserts were present, so maybe the ''lox'' construct is not correct. To check, we will sequence it tomorrow. Afterwards, we miniprepped pC13BF, pC13P, and pC13Q and digested pC13BF. We ran the gel for pC13A and did a PCR clean up for it, as well as for pC13J and pC13L. We also digested pSB1C3 with XbaI and PstI, and miniprepped pC13CV, pC13I, pC13AH and pC13AG. We attempted to run another gel of our PCR product to create Gibson assembly parts, which looked even worse than yesterday. We also did a PCR cleanup of pC13BJ, pC13BK, pC13BL, and pC13BM.<br />
|author=Rafael<br />
}}<br />
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<h3 class="centered">Week 13</h3><br />
<h5 class="centered">(09/09 - 09/15)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 9th<br />
|text= After picking up our pieces from the Clark Hall machine shop, we focused on cutting our insulation to the proper dimensions. Initially, we tried cutting it with a razor blade, but after some rather sloppy results, we decided we needed a more efficient cutting method. Our next attempt involved heating a thin wire and melting through the insulation. This produced better results than the razor blade, but was way too slow. Finally, we decided it would be best if we just resorted to power tools. We bought a jigsaw from Home Depot and cut the insulation like it was cheese. After, we gazed fondly upon the jigsaw and reminisced about high school woodshop. <br />
|author=Mac<br />
}}<br />
<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 9th<br />
|text= PpelA preps were quantified and the <i>G. lucidum</i> promoter was amplified and gel screened. We are preparing our pC13CO, pC13BA, and pC13CL cultures to sequence. The correct insert is in pC13BA based on a gel screen. We digested pC13L and dephosphorylated the pSB1C3 digest from yesterday. We prepared an overnight culture of <i>Cochliobolus</i> and necessary solutions for a protoplasting attempt tomorrow. <br />
|tech= We ran a digest screen on pC13F with KpnI (mRFP has 2 internal KpnI cut sites). No digestion occurred since the enzyme was degraded. We submitted sequencing for pC13CK and pC13BN. We miniprepped pC13CO, pC13BA, pC13CL cultures and need to quantify and prep for sequencing. We ran a colony PCR on streaked pC13BA and pC13C plates and got a definite correct insert on pC13BA, and a lot of nonspecific amplification on pC13CL. We will run a digest screen and sequence. pC13BF colonies were quantified and gel screened and ''PgpdA'' was amplified from <i>G. lucidum</i> DNA, digested, and gel screened. The results were good, thus it will be Gibson assembled. We redid the PCR for pC13A with new materials but it unfortunately failed again. We also digested pC13L with XbaI and PstI and dephosphorylated the pSB1C3 digest from yesterday. We made an overnight culture of <i>Cochliobolus</i> in the Turgeon lab, as well as preparing fresh solutions of enzyme osmoticum and regeneration medium. We miniprepped the cultures of pC13BC, pC13BV, and pC13CE from the weekend. We added antibiotics to CYM plates, and intended to inoculate them for the growth experiment for testing <i>G. lucidum</i>’s antibiotic sensitivity, but the plates were too wet for reliable inoculation (that would spread in a circle of constant diameter), and inoculation was postponed so the plates could dry.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 9th<br />
|text=We met with our design master Oat to discuss design considerations for the presentation. Oat will be making many designs, including a kill switch diagram, homologous recombination figure, our carotenoid image, a toolkit representation, and of course lots of mushrooms. <br />
|tech= <br />
|author=Eric<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 10th<br />
|text= ''Anderson promoter'' + ''T7 polymerase'', in pSB1C3 and BBa_J61002 were miniprepped, glycerol stocked, and prepared for biobrick submission. For fluorescent constructs, a Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. The pC13BN construct was incorrect. An overnight culture was prepared for ''rbs'' + ''crtI'' (from pC13L) at 4C. We made our first attempt at protoplasting and transforming <i>Cochliobolus</i> with the help of the Turgeon Lab. We set up cultures of pC13X and pC13AV in preparation for part submission. We started the growth experiment for testing <i>G. lucidum</i>’s antibiotic sensitivity.<br />
<br />
|tech= We prepared pC13CO, pC13CL, pC13BA for sequencing. We made glycerol stocks of pC13CL and pC13BA. We got a sequence confirmation for pC13CK. We got sequencing for pC13BN and had a portion of the lac operon inside the prefix and suffix...a weird recombination must have occurred. pC13AZ sequencing results were good! That and pA13AM and pA13AW were miniprepped for glycerol stocks and biobrick submission. A PCR to add homologous regions to pC13F for Gibson assembly was done. The gel of Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. Thus, we miniprepped the colonies with correct plasmid insert lengths and submitted them for sequencing. We ran a PCR and electroporation for pC13CV and pC13I, which turned out to be bad. We made overnight E. Coli culture for pC13AF (colony #2). An overnight ligation was done for ''rbs'' + ''crtI'' (from pC13L) at 4C. We ran a PCR to check the inserts in pC13A, pC13I and pC13J. We protoplasted and transformed <i>Cochliobolus</i> with Dongliang, a post-doctoral research associate in the Turgeon Lab. We used our constructs of pAh13G / pC13BR linearized with SphI, as well as the highly efficient <i>Cochliobolus</i> vector PNG from the Turgeon lab as a "positive" control. We submitted pC13BM 1 and pC13BK 1 for sequencing. We homogenized a dilute culture of <i>G. lucidum</i> and inoculated each of the plates for the antibiotic sensitivity test with 20 μL aliquots, without spreading.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 10th<br />
|text=Oat, Eric, Arun, and Hannah sat down to make figures for the presentation. Arun also chose his first color scheme: a baby blue flanked by a serene green. We got many designs done including the carotenoids, homologous regions, kill-switch, and the construct pieces.<br />
|tech=<br />
|author= Arun, Eric<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 11th<br />
|text= Redid transformations of ''PpelA'' and the ''Anderson promoter+T7 polymerase''. We sent the ''Anderson promoter'', pC13CO, pC13CL, and pC13BA to sequencing. Gel electroporation for pC13I showed that it was good, but nothing for pC13J. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists in iGEM! We continued with protoplasting <i>Cochliobolus</i> in the Turgeon Lab, and we ran a Gibson assembly of homology regions in <i>G. lucidum</i>.<br />
<br />
|tech= We submitted pC13CO, pC13CL, and pC13BA for sequencing. After, we miniprepped and made stocks of pC13AY and pC13AZ. Also, we submitted pA13AW for sequencing. Transformation of pK13AX was unsuccessful because the wrong resistance plate was used. pC13BF and pK13AX were transformed today. We miniprepped the overnight culture of pC13AF. Gel electroporation showed that pC13I samples are good, but none of the pC13J samples are positive. We ran a PCR for the inserts in pAK13CR. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists! We made 1000x hygromycin and 100x geniticin. We checked plates from our protoplasting and transforming attempt under the microscope. It appeared unsuccessful so next time we will use all the protoplasts, at least 300 µL. We plated a layer of cym agar and the respective antibiotic on top of the regeneration agar gel as well as inoculating 4 new plates of <i>Cochliobolus</i>. We also ran our Gibson assembly of homology regions in <i>G. lucidum</i>. We miniprepped pC13X and pC13AV in anticipation of part submission, and ran a PCR (to check for inserts) from minipreps of pC13BC, pC13BV, and pC13CE, as well as pC13X and pC13AV (just in case).<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 12th<br />
|text= Ran Gibson assembly for the <i>G. lucidum</i> promoter into pSB1C3. pC13BA is now a BioBrick! We also began cloning a construct with both fluorescence and resistance genes by ligating pC13BR and pC13AT. We successfully electroporated ''E.Coli'' with pC13J! We digested pAK13CR, and then we ligated ''rbs'' + ''crtB'' into pSB1C3, ''crtE'' into pSB1C3. After finding our Gibson assembly from last night left in the water bath, we attempted it again as well as preparing some of our Biobricks for submission. We checked pC13BC, pC13BV, and pC13CE and prepared the successful constructs for sequencing.<br />
<br />
|tech= We prepped pC13BX and pC13BA for BioBrick submission and got sequencing back. pC13BA was correct, and pC13CO and pC13CL were incorrect. We redid the Gibson for pC13CL and transformed it. We redid pC13CO digestions, ligation, and transformations. We transformed the redone ''lox'' Gibson from yesterday. We streaked out CYM plates with <i>Aspergillus Niger</i>, ran the Gibson assembly for ''PgpdA'' in pC13F, and transformed. We also digested pC13BR with SpeI and PstI and digested pC13AT with Xba and PstI, ran a gel of digestions to verify lengths, and column purified the inserts from digestion. We did an electroporation with pC13J and it was successful! We ran a gel for pAK13CR for confirmation and then we did a PCR clean up. We digested pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, and then we ligated ''rbs'' + ''crtB'' (from pAK13CR) into pSB1C3, ''crtE'' (from pC13I) into pSB1C3. We prepared pC13BJ pC13BK pC13BL pC13BM and pC13BR for Biobrick submission. It seems our Gibson assemblies from yesterday were left in the water bath all night, so we reran them (pC13BK + pC13BM + pC13CD, pC13BQ, or pC13Y) before we transformed and plated the new assembled plasmids. We made a culture of pC13CG from glycerol stock. We ran a gel of the PCRs of pC13BC, pC13BV, pC13CE, pC13X, and pC13AV - there were correct bands for at least two of everything, and successful pC13BC, pC13BV, and pC13CE constructs were prepared for sequencing. We also set up a culture of pC13BQ in anticipation of part submission.<br />
<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 13th<br />
|text= Gibson assembly for the <i>G. lucidum</i> promoter yielded no colonies. Numerous ligations and electroporations were done for fluorescent constructs. We are starting the ''afp1'' <i>Aspergillus</i> growth assay. We did electroporation for pC13I, pC13CV and pAK13CS but pAK13CS arced. We prepared numerous Biobricks for submission to iGEM HQ.<br />
<br />
|tech= We submitted pC13BC, pC13BV, and pC13CE for sequencing. We reinoculated cultures of pC13BN, pC13CL, pC13CO. Then, we conducted a preliminary test on activity of ''afp1''. We cut circles of various sizes from dialyzing membranes and dipped in grown ''afp1'' in BL21 culture. The membrane pieces were then placed on the fully grown <i>Aspergillus</i> plate. This preliminary test will see whether or not there is activity of ''afp1'' on the <i>Aspergillus</i>. We are hoping for a ring of death on the <i>Aspergillus</i> plate. Unfortunately, there were no colonies for the ''PgpdA'' assembly. We then ran a gel for some of the remaining assembled DNA and no bands appeared. We also digested pC13AU with Xba and PstI to ligate downstream of pC13BR and pC13BQ. We then column purified pC13BQ, pC13BR, pC13AT, pAK13AO, pC13AU, pC13F. After, we dephosphorylated pC13BQ, pC13BR, pC13F, and ligated pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, and pAK13AO with pC13F. Next, we electroporated all ligations into both BL21 and DH5α cells. Hopefully, a fluorescence trial can be done tomorrow with these colonies! We made overnight culture for pC13J. We did electroporation for pC13I, pC13CV and pAK13CS. The last one unfortunately arced. Our Gibson-transformed plates had too many colonies so we restreaked them. We miniprepped pC13CG and prepared it for Biobrick submission. We also checked our plates in the Turgeon lab. The control plate is growing well, and the others appear to have something very small starting to grow. We will give them more time before checking again. We also received our sequencing results from pC13BM 1, pC13BK 1, pC13BC, pC13BV, and pC13CE, which all appear to have been successful. We set up a culture of pC13CD from its glycerol stock to re-miniprep and submit as a biobrick.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 13th<br />
|text= Managed to get the arduino to control the heating circuit! <br />
|author=Rebecca<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 14th<br />
|text= The ''Anderson promoter'' + ''T7 polymerase'' were electroporated, but both ligations arced, so transformations are very likely to not succeed. We are unsuccessful with pC13CL and pC13BN. We miniprepped pC13J cultures. <br />
<br />
|tech= Electroporated pK13AX, but both arced, so outlook is grim. We also picked 12 colonies from potential pAK13CY colonies and grew corresponding colonies in liquid culture. We then prepped pC13AQ, pC13AT, and pC13AU for biobrick submission. Later that day, we picked colonies for pC13BN and pC13CL from plates from Thursday, did a colony PCR, and grew cultures for each. We re-electroporated the pC13CO construct, and plated four different versions of it. We ran a gel of all 14 colonies of pC13BN and pC13CL. The pC13CL one came out blank and the pC13BN had bands, but not where they were expected. We miniprepped pC13J and made glycerol stock. We ran PCR to check the following inserts: pC13AF, pC13P, pC13CV, pC13I and pC13BG. We ran colony PCRs on the restreaked plates containing our Gibson assembly products of homology regions. We miniprepped pC13BQ and pC13CD for part submission.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 15th<br />
|text=We once again got brunch and ate some school famous chocolate peanut butter cake. We agreed that Becker brunch has the best brunch. Then we sat down all day and put together the majority of the presentation including graphics and content. In addition, Hannah and Eric started writing the script for their part of the presentation. Arun will start writing his script as soon as characterization data starts coming in.<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 15th<br />
|text= We are continuing cloning of ''Lambda Holin'', ''chitinase'', and the ''lox'' sites and adding homologous regions to the ''lox'' sites for Gibson. We started the ''afp1'' assay with <i>Aspergillus</i>. We did a digestion for pC13AF and pC13P, and redid the ligation for insert in pC13L and vector pSB1AK8.<br />
<br />
|tech= We plated 100μL of ''afp1'' culture on fresh <i>Aspergillus</i> plates to demonstrate its effectiveness. We ran a PCR for pC13AI with primers 45/52 and 48/51 to add homologous regions for Gibson. The PCR protocol was a 1-step PCR with a 60C annealing temperature and 1:10 extension using GC buffer. We picked colonies from new pC13CO plates. We miniprepped pC13CL colonies 3 and 4, and pC13BN colonies 5 and 6 although the results of the gel was not clear. We redid the PCR for pC13BN. We also redid the PCR to append cut sites to pC13F for creation of pC13BF. We did a PCR clean up for the inserts in pC13BG, pC13P and pC13AF. We ran a PCR for pC13J and pC13BG. We prepared a double digestion for pC13AF and pC13P, and redid the ligation of ''rbs'' + ''crtI'' (from pC13L) and vector pSB1AK8. We ran gel of the colony PCR of our Gibson products, looking 4-5kb fragments. It appears that pC13BK + pC13BM + pC13BQ colony 1 was the only possibly successful reaction, so we made cultures of it, as well as various other cultures. We prepared pC13BC, pC13BQ, pC13BV, pC13CD, and pC13CE for part submission.<br />
|author=Rafael<br />
}}<br />
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<h3 class="centered">Week 14</h3><br />
<h5 class="centered">(09/16 - 09/22)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 16th<br />
|text=Compared with last week’s jigsaw induced excitement, this week was relatively tame. With all of the necessary pieces in our possession, we set out to assemble our incubator. After sifting through the leftover drylab supplies from last year’s project, we came up with a couple tubes of silicone adhesive that we could use. Insulation pieces were adhered to their respective plastic backings and were allowed to dry for a couple days. <br />
|author=Mac<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 16th<br />
|text=We started taking measurements of <i>G. lucidum</i> growth on CYM plates by tracing the central mass of fungus--the differences between the plates are really interesting! Sequencing for pC13AY was successful so we prepared more culture. We did gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG, but only pC13J colonies are positive. We then did digestions for the following vectors: pC13BG, pC13AG, and pC13KI. We still do not have any growth on antifungal plates.<br />
|tech= We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of <i>G. lucidum</i> growth on CYM plates (plated on September 10) through tracing the central mass of fungus--the differences between the plates are really interesting, but there’s a small contamination on one plate. We transplanted mycelia from the G418 plates. We are slightly concerned that there’s still no growth on the antifungal plates. We submitted pC13CL and pC13BN for sequencing. We ran the gel for the colony PCR of pC13CO but they all failed. We will repick the white colonies from pC13CO plates. We had successful sequencing for pC13AY and we made cultures for glycerol stocks. We also retired the PCR to append cut sites to pC13F using a higher concentration of dNTPs and began a digestion of pA13BE in order to gel extract ''PpelA''. We electroporated ''E. Coli'' with pAK13CS, which unfortunately arced again… We performed gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG. Only pC13J colonies are positive. We also did digestions for the following vectors: pC13BG with SpeI and PstI, pC13AG with EcoRI-HF and XbaI, and pC13K with EcoRI-HF and XbaI.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 17th<br />
|text=We tried transformation of <i>Cochliobolus</i> again. We ran a PCR to append cut sites to pC13F but no band was shown on a gel. We redid the Gibson of ''Cht1_2'' and pSB1C3. When we ran a PCR fo pC13AI, weird band patterning was observed. We purified the vectors we digested yesterday: pC13BG, pC13AG and pC13K. In addition, we purified inserts of pC13AF and pC13P. We continued recording measurements of <i>G. lucidum</i>’s growth in the presence of antibiotics.<br />
<br />
|tech=We attempted transforming <i>Cochliobolus</i> with pC13BR, pC13CD, pUCATPH and pC13AY. We ran a gel of the pA13BE digest and extracted ''PpelA''. We also reran a PCR to append cut sites to pC13F again using a different tube of dNTPs and re-diluted primers. We ran a colony PCR and screened it on a gel, but no bands appeared. Some of the cultures were miniprepped anyways, and they will be screened for presence of the insert (~340bp). We detected some growth on the afp1 assay plates. We redid the Gibson of ''Cht1_2'' and pSB1C3 PCR. We electroporated this Gibson into DH5a cells and plated it on chloramphenicol LB plates. We reran the PCR of pC13AI with primers 45/52 and 48/51, annealing temperature of 61.5C and extended for 30s. We did a double digest of pC13CO with EcoRI-HF & PstI. We ran a 1% TAE gel of pC13AI PCR products for 30 minutes at 100V. The bands for pC13AI with 45/53 were seen at 1.5kb and 1.1kb; bands for pC13AI with 48/51 were seen at 1.5kb and 1kb. We still have a weird banding pattern. We may need to do a gradient PCR to find a better temperature for the primers and to test the PCR without the GC buffer. We purified and dephosphorylated the vectors we digested yesterday--pC13BG, pC13AG and pC13K. We also purified inserts of pC13AF with EcoRI-HF and SpeI, and pC13P with EcoRI-HF and SpeI. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 17th<br />
|text=We met for our first run through of our presentation. The script needs some work, but it’s coming along nicely. It seems that the only part lacking in the presentation is the characterization data and charts. They coming soon!<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 18th<br />
|text=We are getting more data with regards to fungal growth on resistance plates! We ran the pC13CO digests on a gel and they looked good. We ran a PCR of pC13AI to amplify ''lox'' sites for Gibson.<br />
|tech=We used less hygromycin for transformation because Cochliobolus concentrations are less than <i>G. lucidum</i> concentrations. We are recording more and more data with regards with fungal resistance! We found a new contaminant on another plate but we will keep an eye out for it and make sure our measurements will not include its small area of contact with <i>G. lucidum</i>. We ran the gel extracted ''PpelA'' and pC13F PCRs on a gel but there were no bands. However, they may still be successful as the concentration were fairly low and may not have appeared on the gel. We ran the pC13CO digests on the gel and looked good. We ran a PCR of pC13AI to amplify ''lox'' sites for Gibson with harsh conditions to remove the nonspecific amplification. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 19th<br />
|text= Something may be wrong with the pC13AI plasmid based on gel screens. We ligated the constructs from Tuesday--''crtB'' with ''crtI'' and ''crtI'' with PtrpC. We continued recording measurements of <i>G. lucidum</i>’s growth in the presence of antibiotics.<br />
|tech= We quantified the holin minipreps. Both were around 48ng/uL, which is too low to send to sequencing. We will reculture and miniprep when LB is ready. We ran a PCR of pC13AI with primers 45/52 and 48/51. We ran the PCR products on a 1% gel and found bands at 1.5kb and 1.2kb for ''lox1'' and 1.5kb and 1kb for ''lox2''. There may be something wrong with the pC13AI plasmid. We also ran a 1 step PCR of ''lox1'' and ''lox2'' with a 59C annealing temperature. We submitted pC13J for sequencing with Primer 31. We also ligated the constructs from Tue--in short, ''crtB'' + ''crtI'', ''crtI'' + PtrpC. We ran a PCR with Q5 polymerase for inserts in pC13M, pC13J and pAK13CR and ran a gel for them--pC13J was good, pC13M had a faint band but nothing for pAK13CR. We took photos of the plates for the experiment on <i>G. lucidum</i>’s growth in the presence of antibiotics, to capture the differences in appearance in addition to colony size.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 20th<br />
|text= We will start a new growth assay to test afp1. We ran a Q5 PCR for pAK13CR.<br />
|tech= We began another growth assay because the previous ones showed no results. The cells may bave been dead by the time the cells reached them. This method of spreading the ''afp1'' culture like an antibiotic and plating mycelium smoothie on the top should give more consistent results. We retried electroporation with pAK13CS and it sadly arced again… We also ran a Q5 PCR for the insert in pAK13CR. We prepared and autoclaved CYM + agar, for making plates.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 21st<br />
|text= We set up the PCR to append KpnI and BamHI cut sites to pC13F again with different annealing temperatures and ran a gel to test the PCRs. Ran Gibson for pC13AI. A large Q5 PCR was done on fluorescent constructs pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. We ran the gel for pAK13CR from yesterday. We ligated the following: ''RBS'' + ''crtE'' to make pAK13CQ, ''RBS'' + ''crtB'' to make pC13CV, ''ctrB'' + pC13BG to make pC13DP, ''RBS'' + ''crtI'' to make pAK13CS. We attempted to transform ''pC13BV'' into BL21 <i>E. coli</i>, but failed. We then poured CYM plates.<br />
|tech= We ran a Gibson of the DpnI digest of pC13AI PCRs. In terms of characterization, we ran a huge Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F using standard biobrick forward and reverse primers 23 and 24. We set up the PCR to append KpnI and BamHI cut sites to pC13F again with different annealing temperatures and ran a gel to test the PCRs. We ran a gel for the PCR products--insert of pAK13CR--from yesterday, and then we did a PCR clean up of the inserts of pAK13CR and pC13I. We then did electroporation for pC13DJ and pC13BD. The latter arced... We digested pSB1C3 with EcoRI-HF and PstI, pSB1C3 with XbaI and PstI, pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, pSB1AK8 with SpeI and PstI, pC13J with XbaI and PstI, pC13M with XbaI and PstI. We then dephosphorylated digested pSB1C3 and pSB1AK8. Ligation was performed for the following: ''RBS'' + ''crtE'' (from pC13J) into pSB1AK8 to create pAK13CQ, ''RBS'' + ''crtB'' (from pAK13CR) into pSB1C3 to create pC13CV, ''ctrB'' (from pC13M) into pC13BG to create pC13DP, ''RBS'' + ''crtI'' (from pC13L) into pSB1AK8 to create pAK13CS. We transformed ''T7'' + ''hph'' (pC13BV) into BL21 cells and plated on chloramphenicol plates. Unfortunately they arced, so we will need to repeat that with clean electroporation cuvettes. We poured more CYM plates.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 21st<br />
|text=We ran through our slides for the full team. We received a lot of good advice, and we still have a lot of work to do, but most of it will have to wait until after tuesday so we don’t fail our orgo exam! The main suggestion would be to change the theme of the presentation as the baby blue and serene green are too subtle to captivate attention. In addition, there are little glitches to the images.<br />
|tech=<br />
|author=Arun , Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 22nd<br />
|text= We plated pC13AI. We are continuing <i>Aspergillus</i> growth assay, and completed the <i>G. lucidum</i> growth assay.<br />
|tech= We did a PCR cleanup of the pC13AI Gibson pieces, electroporated (time constant was 2.88 ), and plated the cells. We continued taking masses for <i>Aspergillus</i> with ''afp1'' growth assay and began a growth assay with inoculating discs. We measured the colonies of <i>G. lucidum</i> (control, and in the presence of various antibiotics) one final time, as they reached the maximum possible sizes for the petri dishes they were growing in.<br />
|author=Rafael<br />
}}<br />
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<h3 class="centered">Week 15</h3><br />
<h5 class="centered">(09/23 - 09/29)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 23rd<br />
|text= This week, the incubator box was completed. The fabrication and electrical groups came together to begin combining and testing. Initial tests with the temperature sensor yielded very positive results. We were able to successfully set and reach a target temperature with our sensor/feedback system. Unfortunately, our humidity sensor malfunctioned while we were running tests, however, we were still able to gather some data. Initial humidity tests indicate that our misting apparatus is able to produce a 12% increase in humidity over a time span of 8 minutes. Using a hotter water supply, as well as allowing the misting apparatus to run for longer would certainly allow us to increase humidity even more. <br />
|author=Mac<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 23rd<br />
|text= Today we ran a gel to verify insert lengths from Q5 PCR products of fluorescent constructs. We also transformed our T7 + hygromycin resistance construct into <i>E. coli</i> for characterization, and set up a zone of inhibition experiment to test the efficacy of antibiotics and antifungals on <i>G. lucidum</i>.<br />
|tech= Two gels, each with 20 wells, were run simultaneously for fluorescence. The DNA samples loaded were from a Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. pC13BV was transformed into electrocompetent BL21 <i>E. coli</i>. A culture of <i>G. lucidum</i> was diluted and homogenized, and 150 μL aliquots were spread on plates and let dry. Paper discs were soaked in 100X solutions of geniticin, hygromycin, ampicillin, kanamycin, and chloramphenicol and placed in the center of each plate. We miniprepped ''holin'' 8 & 9. The results from the growth assay do not look good. However, we found that the T7 polymerase in BL21 is controlled with an IPTG inducible promoter, so more pC13BA was grown with IPTG and plates were all re-inoculated with the corresponding amounts. An appropriate volume of LB was also added to some plates to account for mass changes. The zone of inhibition tests were also restarted with recultured (with IPTG) pC13BA. The pC13BN transformations yielded no colonies.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 24th<br />
|text=The gel of pC13AI showed nothing - the EtBr (for gel staining) needs to be replaced. We also continued putting our T7+resistances constructs into <i>E. coli</i> for part characterization.<br />
<br />
|tech=We miniprepped the pC13AI cultures. We amplified with primers 45-52 and primers 48-51 and ran a gel (expected size at 1.5kb). Nothing appeared on the gel, not even the ladder. The EtBr should be replaced. There were too many transformants for pC13BV in the BL21 strain, so those cells were restreaked onto a new plate. Additionally, we transformed pC13BC into BL21 as well, and characterize that part as well.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 25th<br />
|text= Sequencing of pC13CO did not turn out to be positive. Still trying to get data from ''Aspergillus'' and growth assay, and characterizing the ''T7 promoter'' and antibiotic resistances constructs. <br />
|tech= The sequencing of pC13CO came back as ''PtrpC'' + ''GFP''. We are not sure why or how. We either miniprepped the wrong thing or put the wrong thing into sequencing. We continued to take measurements for growth assays. We put a lot of <i>Aspergillus</i> around the inoculating discs for gathering more data. We set up cultures of BL21, with no plasmid and with pC13BC and pC13BV, and then standardized the optical densities of the cells between the three cultures. We spread 50 μL aliquots onto LB, adding discs dipped in 10X, 1X, and 0X concentrations of geneticin and hygromycin.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 25th<br />
|text=We made a lot of changes to the presentation tonight, and it is an almost polished form now. Arun switched to his next color scheme: a deep Cornell red with baby blue banners. Eric has gotten his part of the script memorized. He will be speaking about the three components of our project. The fungal toolkit, and its implementation through the carotenoid pathway and antifungals expression. Hannah has also prepared her part on human practices. In addition she has prepared an analysis on the market trends towards bioplastics.<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 26th<br />
|text=The ''T7 promoter'' and hygromycin resistance construct appears functional in <i>E. coli</i>.<br />
|tech=There was a clear zone of inhibition around the discs dipped in 10X hygromycin, no inhibition around the discs dipped in sterile water, for the wild type BL21 <i>E. coli</i>. However, there was normal growth around the 10X hygromycin discs for the BL21 <i>E. coli</i> transformed with our pC13BV construct. For the geneticin tests, the antibiotic appeared relatively ineffective, possibly due to degradation, and that experiment will need to be repeated.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 26th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. <br />
|tech=<br />
|author=Eric<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=pres<br />
|head=September 27th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. Arun also experimented with backgrounds of clouds, dirt, and toolkit to make the introduction slides very sharp. It look like we now have a solid design for our presentation.<br />
|tech=<br />
|author=Arun,Eric<br />
}}<br />
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<h5 class="centered">(09/30 - 10/06)</h5><br />
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<h5 class="centered">(10/07 - 10/13)</h5><br />
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<h5 class="centered">(10/14 - 10/20)</h5><br />
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<h5 class="centered">(10/21 - 10/28)</h5><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:CornellTeam:Cornell2013-09-28T04:13:59Z<p>R.Lizarralde: Undo revision 243134 by R.Lizarralde (talk)</p>
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We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.<br />
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Notebook entries for all activities from Wetlab, Drylab, Human Practices, Wiki, and Presenters.<br />
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Meet the brains and personalities behind this year's project.<br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><img src="https://static.igem.org/mediawiki/2013/1/17/Outreach.png"></a><br />
</div><br />
<div class="seven columns homepage-force"><br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><h3 style="margin:0px auto 0px;">Outreach</h3></a><br />
</div><br />
</div><br />
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Educating the community on our research and understanding the public opinion of synthetic biology.<br />
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<a href="https://2013.igem.org/Team:Cornell/outreach" class="small button radius corner">More Info</a><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:CornellTeam:Cornell2013-09-28T04:11:48Z<p>R.Lizarralde: </p>
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<div class="row"><br />
<div class="twelve columns"><br />
We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.<br />
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Notebook entries for all activities from Wetlab, Drylab, Human Practices, Wiki, and Presenters.<br />
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Meet the brains and personalities behind this year's project.<br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><img src="https://static.igem.org/mediawiki/2013/1/17/Outreach.png"></a><br />
</div><br />
<div class="seven columns homepage-force"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<a href="https://2013.igem.org/Team:Cornell/outreach"><h3 style="margin:0px auto 0px;">Outreach</h3></a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="twelve columns"><br />
Educating the community on our research and understanding the public opinion of synthetic biology.<br />
</div><br />
</div><br />
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<a href="https://2013.igem.org/Team:Cornell/outreach" class="small button radius corner">More Info</a><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:CornellTeam:Cornell2013-09-28T04:11:33Z<p>R.Lizarralde: Undo revision 243082 by R.Lizarralde (talk)</p>
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<style type="text/css"><br />
img#igem-attr {<br />
transition: transform 0.5s;<br />
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}<br />
<br />
img#igem-attr:hover {<br />
transform: rotate(-15deg);<br />
-ms-transform: rotate(-15deg); /* IE 9 */<br />
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<div class="row"><br />
<div class="twelve columns"><br />
<a href="https://2013.igem.org/Team:Cornell/project"><h3 style="margin:0px auto 0px;">Project</h3></a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="twelve columns"><br />
We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.<br />
</div><br />
</div><br />
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<a href="https://2013.igem.org/Team:Cornell/project" class="small button radius">More Info</a><br />
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Notebook entries for all activities from Wetlab, Drylab, Human Practices, Wiki, and Presenters.<br />
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<a href="https://2012.igem.org/Team:Cornell/team"><h3 style="margin:0px auto 0px;">Team</h3></a><br />
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</div><br />
<div class="row"><br />
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Meet the brains and personalities behind this year's project.<br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><img src="https://static.igem.org/mediawiki/2013/1/17/Outreach.png"></a><br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><h3 style="margin:0px auto 0px;">Outreach</h3></a><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="twelve columns"><br />
Educating the community on our research and understanding the public opinion of synthetic biology.<br />
</div><br />
</div><br />
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<div class="twelve columns controls" ><br />
<a href="https://2013.igem.org/Team:Cornell/outreach" class="small button radius corner">More Info</a><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:CornellTeam:Cornell2013-09-28T04:10:47Z<p>R.Lizarralde: </p>
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-o-transition: -o-transform 0.5s; /* Opera */<br />
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img#igem-attr:hover {<br />
transform: rotate(-15deg);<br />
-ms-transform: rotate(-15deg); /* IE 9 */<br />
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<a href="https://2013.igem.org/Team:Cornell/project"><h3 style="margin:0px auto 0px;">Project</h3></a><br />
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We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.<br />
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<a href="https://2013.igem.org/Team:Cornell/project" class="small button radius">More Info</a><br />
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<a href="https://2013.igem.org/Team:Cornell/notebook"><img src="https://static.igem.org/mediawiki/igem.org/2/25/Cornell_IMG_5012.JPG"></a><br />
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<a href="https://2013.igem.org/Team:Cornell/notebook"><h3 style="margin:0px auto 0px;">Notebook</h3></a><br />
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Notebook entries for all activities from Wetlab, Drylab, Human Practices, Wiki, and Presenters.<br />
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<a href="https://2013.igem.org/Team:Cornell/notebook" class="small button radius corner">More Info</a><br />
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<a href="https://2013.igem.org/Team:Cornell/team"><img src="https://static.igem.org/mediawiki/2013/1/19/Team_cornell_2013.png"></a><br />
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<a href="https://2012.igem.org/Team:Cornell/team"><h3 style="margin:0px auto 0px;">Team</h3></a><br />
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Meet the brains and personalities behind this year's project.<br />
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<a href="https://2013.igem.org/Team:Cornell/team" class="small button radius corner">More Info</a><br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><img src="https://static.igem.org/mediawiki/2013/1/17/Outreach.png"></a><br />
</div><br />
<div class="seven columns homepage-force"><br />
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<a href="https://2013.igem.org/Team:Cornell/outreach"><h3 style="margin:0px auto 0px;">Outreach</h3></a><br />
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Educating the community on our research and understanding the public opinion of synthetic biology.<br />
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<a href="https://2013.igem.org/Team:Cornell/outreach" class="small button radius corner">More Info</a><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/File:MycoGEMSlider.gifFile:MycoGEMSlider.gif2013-09-28T04:10:31Z<p>R.Lizarralde: </p>
<hr />
<div></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/templates/headerTeam:Cornell/templates/header2013-09-28T03:55:32Z<p>R.Lizarralde: </p>
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<a href="https://2013.igem.org/Team:Cornell/project">Project</a><br />
<a href="https://2013.igem.org/Team:Cornell/project" class="flyout-toggle"><span> </span></a><br />
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<a href="https://2013.igem.org/Team:Cornell/project">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background">Background</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab">Dry Lab</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/hprac">Human Practices</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/future">Future Applications</a><br />
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<a href="https://2013.igem.org/Team:Cornell/notebook">Notebook</a><br />
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<a href="https://2013.igem.org/Team:Cornell/notebook">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/notebook/protocols">Protocols</a><br />
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<a href="https://2013.igem.org/Team:Cornell/notebook/plasmids">Plasmids</a><br />
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<a href="https://2013.igem.org/Team:Cornell" style="margin-top:-50px">2013: Organofoam</a><br />
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<a href="https://2010.igem.org/Team:Cornell">2010: OMG OMVs</a><br />
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<a href="https://2009.igem.org/Team:Cornell" style="font-size:20px !important">2009: Cadmium Sensor</a><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/notebookTeam:Cornell/notebook2013-09-28T03:53:38Z<p>R.Lizarralde: </p>
<hr />
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<!-- -------------------Notebook entries------------------- --><br />
<div id="weekAentry" class="nb-week" style="display: block"><br />
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<h3 class="centered">January</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 5th<br />
|text=Applications for new Cornell iGEM members were due today-- we've got 57!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 16th<br />
|text=After an eternity of reading and a gigantic spreadsheet of evaluations, we're finally ready to start interviewing applicants-- 36 of them!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 25th<br />
|text=I'm really excited about having these new members on the team. There were many awesome interviews, but now we have to sit down and make hard decisions on who to take, as most of last year's members are graduating, so there's a limit to how many new members we can train. This is going to be a tough weekend.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 27th<br />
|text=After several hours of discussion, we decided on our 21 new members! Congratulations, Tim Abbott, Hannah Ajmani, Eric Appel, Ryan Ashley, Nupur Bhatt, Arun Chakravorty, Rebecca Chew, Sharlene Dong, Sara Gregg, Alex Han, Eric Holmes, Daniel Leach, Oat Luengvarinkul, Jeffrey Ly, Ritvik Sarkar, Mac Sennett, Prashant Sharma, Olya Spassibojko, Yoshiko Toyoda, and Kyle Wheeler!<br />
|author=Rafael<br />
}}<br />
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<h3 class="centered">February</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 1st<br />
|text=We had our first full team meeting today with all the new members, introducing them to the iGEM competition, and did an icebreaker we call speed-taboodunit! For the next week, they have the assignment of analyzing a previous iGEM project (in groups), and explaining what the project was trying to accomplish, whether synthetic biology was a good approach for this, and reasons for their success or lack thereof.<br />
|tech=Speed-taboodunit is a mashup of speed-dating, taboo, and whodunit; the idea is that each person writes on a piece of paper something unusual about themselves, underlining the key words, then these pieces of paper are randomly redistributed. Everyone organizes in a speed-dating format, with two lines of people, and you have a minute to talk to them and try to find out if they're the person described on their piece of paper-- with the catch that they can't use any of the underlined words. People who successfully find their person leave the group, but otherwise after the minute everyone moves down the line. As for why we came up with this specific icebreaker, we wanted to hit two key requirements of icebreakers: they have to be fun, and they have to make specific accommodations for being shy or quiet. Because each person has the specific objective of finding their person, this helps them overcome the shyness, and as for the fun, everyone loves taboo!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 7th<br />
|text=Our first social event tonight went well! Hopefully with a few more of these we'll have some great team comradery and feel more comfortable around each other. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 9th<br />
|text=At our second team meeting we went over the past project analyses, as well as some biology review-- mostly molecular cloning. For the next week, we've assigned more past projects for them to outline in more detail.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 16th<br />
|text=Today we went over literature review, went over the past project outlines, and gave a two-week problem-oriented brainstorming and project outline assignment. Now that each member has looked at several past projects and analyzed them, we're moving into brainstorming for this year's project and practicing the development of project pitches.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=February 21th<br />
|text=We just started working on the wiki! Our first assignment: figuring out how wiki templates work. With templates the website would become much easier to edit, but for some reason HTML and wiki text don't seem to work very well together...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 23th<br />
|text=For the second week of this assignment, we compiled the brainstormed ideas and had members pick which ones they wanted to explore further. We also had a movie night planned, but it fell through :(<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=February 28th<br />
|text=Finally got templates to work! The trick was ending HTML right before wiki text, and starting HTML again right afterwards. Not very elegant, but it works.<br />
|author=Nupur<br />
}}<br />
<html> <br />
</div> <br />
</div><br />
<div id="weekCentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">March</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 1st<br />
|text=We want to revamp the notebook page with a filter system that lets you sort by subteam. We set up the basic layout of the page, but writing the javascript is going to be another story.<br />
|tech=Check out our <html><a href=https://2012.igem.org/wiki/index.php?title=Team:Cornell/Sandbox/notebook&oldid=299789 target="_blank"> notebook</a></html> before anything worked!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 2nd<br />
|text=Groups over the previous week investigated phage therapy, piezoelectric bacteria, "sticky paper", logic gates / bistable switches, phytases, mycelia, and pressure-adaptive materials. We're keeping the results from those, but doing a second round of brainstorming over the next few weeks-- the idea is to allow the exploration of any ideas that arose while researching the previous round of ideas.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 5th<br />
|text=We just finished the javascript for filtering. Rafael also made cool subteam icons for the filter buttons.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 6th<br />
|text=I made a template for notebook entries and added styling to each entry.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 9th<br />
|text=We compiled our second round of ideas, including topics like methanotrophs, a CRISPR knockout system, and suppressor tRNA inducible expression systems. We also took our first team picture, and probably the only one that will have all of last year's and this year's members together!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 29rd<br />
|text=Spring break knocked out a couple of meetings, but it looks like some research was accomplished nonetheless! The phytase project idea is looking pretty promising-- it incorporates a lot of different disciplines, with stuff for our mechanical and electrical engineers to do, as well as modeling.<br />
|tech=A lot of iGEM teams we've looked at don't have much beyond the biology, so we really try hard to do interdisciplinary projects, as they provide a great opportunity for learning to work with different kinds of people.<br />
|author=Rafael<br />
}}<br />
<html> <br />
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</div><br />
<div id="weekDentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">April</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 6th<br />
|text=We've narrowed it down to four project ideas: smokestack reactors (capturing pollutants with bioreactors), incorporation of phytases into anaerobic digesters, mycelial materials, and using bacteria to address issues with artificial implantable kidneys.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 10th<br />
|text=We had a call today with Ecovative Design, a company that uses mushrooms to make a biodegradable styrofoam analogue-- our mycelial materials project idea would involve working with them to improve their product. It seems like our interests are aligned and that we should be able to work with them. Exciting! <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 13th<br />
|text=This meeting was dedicated to project pitches from each of the four ideas, followed by voting on which project we should pursue. Some of our graduate advisors also attended to provide a more seasoned perspective on the viability of each project. After some deliberation and spreadsheet magic, we decided on mycelial materials!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 20th<br />
|text=Today we worked on team structure for the summer; Swati will be the overall team leader, with Mark and I coordinating wetlab, Olya taking meeting minutes, Mac running drylab, and Hannah will be managing human practices. Later on in the summer, we will also develop task groups. For the next week, we've also split into groups to research various aspects of the mycelial materials project.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 26th<br />
|text=We've compiled initial research on carotenoid pigments, transformation protocols, DNA that may be useful for a basidiomycete toolkit, and a few other topics. Over the next week we'll meet with several professors on campus who work with fungi to see what resources and advice they can provide.<br />
|author=Rafael<br />
}}<br />
<html> <br />
</div> <br />
</div><br />
<div id="weekEentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">May</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=May 4th<br />
|text=We met with Professor Gillian Turgeon, who does fungal transformation with Cochliobolus heterostrophus, an ascomycotic corn pathogen. We can get a few of the genes we've been looking for from her, yay! We also met with Professor Teresa Pawlowska, who directed us towards some other fungi and resources for fungi, as well as provided some background on fungal biology. As for the rest of the semester, we're letting everyone off the hook-- we don't want anyone to fail their exams because of iGEM!<br />
|author=Rafael<br />
}}<br />
<html> <br />
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</div><br />
<div id="week1entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered" >Week 1</h3><br />
<h5 class="centered">(06/17 - 06/23)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 17th<br />
|text=The drylab team met briefly on June 17 to begin discussing some ideas. We went over some of the growing methods for Ganoderma lucidum, brainstormed some non-packaging related ideas for a fungal biomaterial, and discussed how we could test some of the physical characteristics of the final product. We thought it might be a good idea to test the thermal conductivity if it were to be used as a drinking cup or something. We also determined that people probably do not want to drink out of a fungus cup. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 18th<br />
|text=We started bootcamp today, doing our first transformations with kit plate biobricks.<br />
|tech=We transformed parts containing carotenoid pathway genes ''crtE'', ''crtI'', ''crtB'', and ''crtY'' (BBa_K523022, BBa_K539119, BBa_K145001), as well as the T7 promoter (both in pIVEX2.3d as well as BBa_I712074), the T7 polymerase gene (BBa_K145001), and the transformation efficiency kit part (BBa_J04450).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 19th<br />
|text=The second day of bootcamp had more activity-- we PCR-amplified a variety of parts to biobrick them and get ready for making composite parts later.<br />
|tech=From the plasmid pUCATPH (pAh13G), we amplified the ''trpC'' promoter (P''trpC'', a constitutive fungal promoter) and the ''trpC'' terminator (T''trpC''). From pNG (pAg13H), we amplified ''nptII'', the geneticin (a plant/fungal antibiotic) resistance gene.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 20th<br />
|text=I finally started moving pages to the 2013 wiki! It's looking very empty right now, but Rafael made a gif of our logo with his mycodraw program to use as a homepage placeholder. We also decided to add a technical details section to each of our notebook entries so that we can separate the jargon from daily summaries.<br />
|tech=Check out the <html><a href=https://static.igem.org/mediawiki/2013/2/21/CornellStillGrowing.gif target="_blank">gif</a></html> if it's not on our homepage anymore. Originally Rafael converted his mycodraw program from java to javascript (which took a VERY long time), but the script only worked on some browsers :( We had to settle for the gif instead.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 20th<br />
|text=On our third day of bootcamp, we taught new members how to run gels, do minipreps, and run digests, continuing our preparation of biobrick parts from the previous two days.<br />
|tech=We miniprepped the ''crtEIB'' (BBa_K523022), ''crtY'' (BBa_K539119), and T7 (BBa_I712074) biobricks. The gels confirmed our PCRs for P''trpC'', T''trpC'', and ''nptII'', which we then digested to insert into pSB1C3.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 21st<br />
|text=The javascript for toggling technical details took a bit longer than expected, but we finished it up today.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 21st<br />
|text=Continuing with bootcamp, we column-purified the digests of PCR products and gel-purified the digested pSB1C3 backbone, dephosphorylated, and ligated, resulting in our first batch of biobricks or pre-biobrick parts.<br />
|tech=P''trpC'', T''trpC'', and ''nptII'' were column-purified, digested pSB1C3 backbone was gel-purified and dephosphorylated, and each was ligated into the pSB1C3 backbone.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 22nd<br />
|text=I added a flyout to notebook entries authors with a picture, short description, and link to their bio on the team page.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 22nd<br />
|text=Our penultimate day of bootcamp was a light one-- we only transformed cells with the constructs we'd made over the past four days. <br />
|tech=We transformed T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) constructs into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 23rd<br />
|text=We concluded bootcamp with colony PCRs to test whether each of the constructs was present. Unfortunately, the gels had no bands, so we grew overnight cultures and will screen the minipreps instead.<br />
|tech=We used Q5 PCRs with the standard biobrick primers VF2 and VR to attempt to confirm the presence of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R).<br />
|author=Rafael<br />
}}<br />
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</div><br />
</div><br />
<div id="week2entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 2</h3><br />
<h5 class="centered">(06/24 - 06/30)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 24th<br />
|text=The drylab team met to update each other on some of the preliminary research. The main focus of this meeting was the development of a growing protocol. Sara found a professor that we could contact in the material science department for help regarding the testing of material properties. Rebecca and Manny looked into what different aspects of the fungi we could model, such as growth, decay, etc. Ritvik brainstormed some more ideas regarding the potential uses of this material, and came up with the idea of using it as insulation. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 24th<br />
|text=I started working on a hypertexting script that will automatically recognize words on a page and add a flyout with a description. We can use this to recognize names for the author flyouts I implemented yesterday, as well as other keywords such as plasmid names and protocols.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 24th<br />
|text=We miniprepped our three constructs. Then we went back to sleep.<br />
|tech=Minipreps of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 25th<br />
|text=I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 25th<br />
|text=We did another PCR from the miniprepped plasmids to confirm the right sizes of inserts, then sent them in for sequencing.<br />
|tech=The Q5 PCRs of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) showed bands of the correct size, so sequencing with VF2 and VR was submitted to make sure nothing strange had happened.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 26th<br />
|text=The script is now working on all browsers except for Internet Explorer.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 27th<br />
|text=We started cloning crt genes downsteam of the T7 promoter. Because the T7 promoter is too small to extract as a fragment, we're placing crt genes downstream, then transferring the promoter+gene fragments back to pSB1C3. Today we digested ''crtY'' and the T7 promoter plasmid, but the digestion of ''crtY'' failed. <br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). However, the gel showed only one band for each (what one would expect for pAK13D, but pC13B should have had two bands).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 28th<br />
|text=We redid the digestion of ''crtY'' and the T7 promoter plasmid from yesterday, apparently successfully this time. We also started liquid cultures of pBARGPE1, a plasmid we got from the Fungal Genetics Stock Center.<br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). The gel this time had two bands at the correct locations for pC13B, so we're hoping it's a success this time. We also started overnight LB cultures of pAb13T (pBARGPE1).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 29th<br />
|text=Crossing our fingers, we gel extracted and ligated the ostensibly successful digests of ''crtY'' and the T7 promoter plasmid, and then transformed. We also digested ''crtI'' and ''crtB'', other carotenoid genes that we will also be putting under the T7 promoter, and miniprepped the pBARGPE1 that had been grown up last night.<br />
|tech=If successful, the ligation will form pAK13AD. pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI for insertion into pAK13D, and separated on a gel. pAb13T (pBARGPE1) was miniprepped and quantified by nanodrop. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 30th<br />
|text=Unfortunately, there were no colonies on the transformation plate for ''T7'' + ''crtY'', so we redid the digestions for those. We also digested ''TtrpC'' and ''nptII'' for the eventual cloning of a ''PtrpC''+''nptII''+''TtrpC'' construct (we have to put the parts together in reverse because ''nptII'' contains a PstI site, which we will be trying to remove as well). Gel extractions of digested ''crtI'' and ''crtB'' were also empty (insignificant concentrations), so these digestions were also redone, and all were run on a gel, which looked mostly okay.<br />
|tech=pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (''TtrpC'') with EcoRI and XbaI, and pCg13R (''nptII'') with EcoRI and SpeI, and all were separated on a gel; ''crtI'' looked faint, and pAK13D was smeared, but all fragments were cut out for purification tomorrow.<br />
|author=Rafael<br />
}}<br />
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</div><br />
<div id="week3entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 3</h3><br />
<h5 class="centered">(07/01 - 07/07)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 1st<br />
|text=We finished the hypertexting script and fixed some miscellaneous problems. I think we can call this a wrap. The notebook page is officially done!<br />
|tech=Now we just have to start filling out entries...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 1st<br />
|text= Two of our team members participated in SILS Skills Night on June 20th.<br />
|tech= Price of one 12 oz Styrofoam cup: 4.09 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 1st<br />
|text=Yesterday's digestions were gel extracted, ligated, and transformed. Hopefully it worked this time!<br />
|tech=Digested pAK13D was dephosphorylated, then ''crtI'' , ''crtB'', and ''crtY'' were ligated with pAK13D to put them under the T7 promoter, hopefully producing pAK13AB, pAK13AC, and pAK13AD, respectively. pCg13R was also ligated with pC13Q, hopefully producing pCg13U. All four constructs were transformed into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 2nd<br />
|text=There were no transformants, again :(. On the other hand, we received our tube of <i>Ganoderma Lucidum</i> from Dr. David Hibbett today! Once more with feeling, we redid the four digestion-ligations for ''crtI'', ''crtB'', ''crtY'', and ''nptII''. We also amplified ''hph'', the hygromycin resistance gene, from one of the plasmids we got from Dr. Turgeon's lab, as well as pSB1C3 backbone, and we will use Gibson assembly to put them together (as ''hph'' has both EcoRI and PstI sites)-- the gels looked good for both of these. Finally, we submitted pAb13T (pBARGPE1) for sequencing to determine the sequences of the ''Aspergillus nidulans'' ''gpdA'' promoter and the ''bar'' gene, which confers phosphonothricin resistance.<br />
|tech=''hph'' was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 3rd<br />
|text=All transformations failed yet again, and were repeated once more. Four tears shed for the lost. ''hph'' was Gibson-assembled, and we ran a Q5 PCR of the ''bar'' gene, under the assumption it matches other published ''bar'' sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on ''nptII'' (pCg13R) to remove its internal PstI site.<br />
|tech=:( ;_; T_T D: (tears for the transformations lost). The ''bar'' PCR and pC13F were digested with EcoRI and PstI, and ligated to hopefully form pCp13X. Site-directed mutagenesis was performed both with and without DpnI to digest template DNA. <add something about site-directed mutagenesis design; refer to primers? probably requires Swatinput><br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 4th<br />
|text=The previous days ligations were all desalted and transformed, yet again.<br />
|tech=The previous days ligations were all desalted and transformed, yet again, into E. Coli and plated, including the non-GC trials of ''nptII'' site-directed mutagenesis. Digestions on ''bar'' and pSB1C3 were purified and run on a gel. They appeared successful, and pSB1C3 was gel-extracted for ligation with the PCR-cleanup of ''bar''. The digests of pSB1C3 and ''bar'' were quantified and ligated for submission as a Biobrick. Finally, liquid broth cultures of the hygromycin Gibson transformants were created.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 5th<br />
|text= All of yesterdays transformations failed, in addition to the ''hph'' Gibson :-( The ligase was tested by ligating DNA ladder: All were discovered to be non-functional. Why life? Why?<br />
|tech= The site directed mutagenesis cultures of ''nptII'' were put into liquid cultures from plates. One liquid culture of a transformant of ''crtY'' in pAK13D was also made, along with a reference plate. A ligation of ''bar'' in pSB1C3 was desalted and transformed. Only one of the Gibson assemblies appeared successful, failing to express rfp, and a glycerol stock was made of that culture. After further incubation, the successful culture was miniprepped and PCR'ed.When run on a gel, that culture appeared ineffective.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 5th<br />
|text=Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 6th<br />
|text=The ''crtY'' ligation was run on a gel and shown to be unsuccessful. The ''hph'' PCR also came up blank, so the Gibson was also unsuccessful. On the bright side, a digestion screen on ''nptII'' shows that site-directed mutagenesis on that site most likely worked!<br />
|tech=Colonies of ''nptII'', ''nptII'' digested with DpnI, ''hph'', and ''crtY'' were miniprepped. ''nptII''+ DpnI sample 4 was accidentally pipetted into the sample 3 column, so contamination is possible. Concentrations were good. A digest screen was performed on ''nptII'' using PstI, and ''crtY'' was digested with EcoRI and PstI. Fragments of ''nptII'' digested with DpnI ~2.5kb and linearized mutated ~3kb. The ''nptII'' digested with DpnI appeared successful, while the undigested samples failed. Gels of ''hph'', and ''crtY'' were also run, showing that the ''crtY'' ligations failed, as well as the ''hph'' Gibson.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 7th<br />
|text=A pSB1C3 backbone was prepared for Gibson assembly and gel extracted; however, we later were told by a representative from New England Biolabs that gel extraction might mess up a Gibson assembly, rendering todays extractions useless.<br />
|tech=A Miniprep was run on three ''bar'' cultures in preparation for later ligation into pSB1C3. These were then run on a gel along with Gibson-ready pSB1C3. Although loading dye bubbling out of the wells made it appear as though no results would come of the gel, the gel was successful. ''bar'' was confirmed and a gel extraction was performed on pSB1C3 and frozen.<br />
|author=Kyle<br />
}}<br />
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</div><br />
<div id="week4entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 4</h3><br />
<h5 class="centered">(07/08 - 07/14)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 8th<br />
|text=Several constructs were ligated. The site-directed mutagenesis of ''nptII'' in pCg13S was ligated with ''TtrpC''. Assembled ''bar'' in pSB1C3, submitted for sequencing. The Gibson of ''hph'' into pSB1C3 was rerun. An experimental method of ligating ''crtY'' into pAK13D upstream of the T7 promoter was tried as an alternative to gel purification and PCR purification, since we lack the primers to PCR and gel purification interferes with ligation.<br />
|tech=Digested ''nptII'' in pCg13S with EcoRI+XbaI, and TtrpC with EcoRI+SpeI. The ''crtY'' in pSB1C3 was digested four different ways: One with just XbaI & PstI; one with XbaI, PstI, and SacI; one with XbaI, PstI, and ApaLI; and one with XbaI, PstI, SacI, and ApaLI. Although unlikely, this method is intended to prevent re-ligation of the backbone of ''crtY'' during digestion. More ''crtY'', ''crtI'', ''crtB'' was inoculated in liquid cultures for future miniprepping.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 9th<br />
|text=All yesterdays ligations were dialyzed and plated. The Gibson of ''hph'' into pSB1C3 grew red colonies, showing it was unsuccessful, so the procedure was performed again. The ''bar'' ligation came back successful!<br />
|tech=<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 10th<br />
|text=The ''nptII'' ligation with ''TtrpC'' grew colonies was unsuccessful. In other news, much lab organization was done today!<br />
|tech=The ''nptII''+''TtrpC'' ligation was checked by doing a colony PCR on the ligation colonies and a subsequent gel. Possible gel outcomes were plasmid self ligation (200bp) in which ''TtrpC'' was randomly cut out of pC13Q, 800bp if the plasmid has only ''TtrpC'', ~1kb indicating ''nptII'' noise from PCR template, and ~1.7 kb indicating successful construct formulation. The gel showed two bars at ~800bp, indicating a failed ligation. The third Gibson attempt of ''hph'' into pSB1C3 was also plated, and a 10mL culture was made of the successful ''nptII'' DpnI mutagenesis plate, evidently for future great justice.<br />
|author=Kyle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 10th<br />
|text= Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 11th<br />
|text=After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop. <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 11th<br />
|text=Digest screens of previous ''crtY'' ligations showed promising results. Gibson cloning was continued, and further cloning of ''nptIIBB'', to place it in front of PtrpC, was prepared.<br />
|tech=Minipreps were made from the ''crtY'' plates, from which a digest screen was prepared using NotI to see if the ligation worked. The digestions were run on a gel. Samples with XbaI, PstI, and SpeI; XbaI, PstI, and ApaLI numbers 2 and 3; and XbaI, PstI, SpeI, and ApaLI numbers 2 and 3 all worked. A miniprep of ''nptIIBB'' was performed. The concentration was extremely low, so an overnight culture was made from the glycerol stock to be miniprepped tomorrow. Another transformation was done using remaining previous minipreps of ''nptII BB''. Cultures were made of the third Gibson attempt, and a colony PCR performed. When run on a gel, the non-GC fragments were too small, but GC colonies number 2 and 5 were good, at 1kb, and number 4 had a band at 1kb with another smaller fragment. Finally, ''nptII BB'' was digested with XbaI and PstI, then column purified. PtrpC was digested with SpeI and PstI and gel purified.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 12th<br />
|text=Cloning with ''nptIIBB'' and PtrpC was continued, as well as cloning with the Gibson Assembly method.<br />
|tech=In the morning, the digestions of ''nptIIBB'' and PtrpC were quantified. A miniprep of ''nptIIBB'' transformants was performed to replenish DNA, and another glycerol stock of ''nptII BB'' was made from a leftover culture. The GC colonies 2, 4, and 5 from yesterdays gel were prepared for sequencing. A ligation was performed on ''nptIIBB'' and PtrpC. More cultures of the successful ''crtY'' ligations were made. CYM medium was created, and ''PC13Z'' was transformed from a kit plate.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 13th<br />
|text=Today we worked with fungi! We applied different amounts of antibiotic to G. lucidum, to test the sensitivity of our strain of fungi. In addition, cloning of pCg13Y was nearly completed. <br />
|tech=The ligation of ''nptIIBB'' with PtrpC was desalted, transformed into E. coli and plated. Antibiotic stocks of geneticin and phosphinothricin were made at 100x concentration. <i>G. lucidum</i> cultures were inoculated with varying levels of antibiotic to test the strain's sensitivity for future selection purposes.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 14th<br />
|text=Cloning of ''nptIIBB'' in front of PtrpC was unsuccessful. Plasmids pC13z and pAK13AD were prepped for cloning.<br />
|tech= Colony PCRs and cultures were completed to determine the success of cloning ''nptIIBB'' downstream of PtrpC. Unfortunately, a gel revealed that the dna fragments appeared at 0.6kb, shorter than the expected 1.4kb of a successful ligation. pC13Z and pAK13AD were miniprepped, but had to be thrown out due to contamination of our EB. New cultures were made to be miniprepped again. A glycerol stock of pC13Z was also made.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 14th<br />
|text=This week, drylab split up so members could focus on their strengths. Rebecca and Sara began designing our feedback controller, with some much appreciated advice from the previous drylab team leader, Dan. Ritvik, Manny, and Mac began coming up with design considerations for the incubation chamber. We were most concerned with size; too small and it wouldn't be useful, too big and it would be difficult to control any variables. We eventually decided that a 1'x1'x2' chamber would probably be a good compromise. Rebecca, Sara, Ritvik, and Mac also attended machine shop training this week. In order to complete our training, we each had to construct a small aluminum lamp using a milling machine and a lathe. Fortunately, everyone emerged from the training session one lamp richer, and with a full set of fingers on each hand.<br />
|author=Mac <br />
}}<br />
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<div id="week5entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 5</h3><br />
<h5 class="centered">(07/15 - 07/21)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 15th<br />
|text=Rebecca looked into the components of the temperature feedback control, namely, the temperature sensor, the microcontroller, and the heating element. Put in an order for the temperature sensor, microcontroller (an Arduino Fio), and Xbee ports for wireless communications.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 15th<br />
|text=Gibson ''hph'' was submitted for sequencing, and ALL of the cloning was performed.<br />
<br />
|tech=Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. To begin, the Gibson ''hph'' was submitted for sequencing, and pAK13AD and pC13Z were miniprepped. A PCR of pC13Z, pC13F, pCP13X, pC13M, and pC13E was then run with primers 31 and 32, the standard pSB1C3 primers. A gel was run to check the PCRs. All the PCRs looked successful except pC13M and pC13E, which had nonspecific bands around 1kb. A new technique was used on a few of the digests involving a restriction cocktail of enzymes to cut the DNA. Plasmid pAK13AD was digested with EcoRI, SpeI, SacI, and ClaI so that it could be placed in pSB1C3. We used SpeI instead of PstI due to the small distance between the PstI and the SacI cut sites. Plasmid pC13F was digested with EcoRI, SpeI, and KpnI so pAK13AD could be placed in it. Plasmid pAK13D was digested with SpeI and PstI so ''crtI'' and ''crtB'' could be placed in it. All the digests were then column purified rather than gel purified--a move which has improved our ligation efficiencies. The purifications were quantified. Cultures were made for pC13P and pC13K and, finally, pAK13AD was dephosphorylated then ligated into pC13F (pC13AH).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 16th<br />
|text=One iGEM member spoke at a panel for pre-freshmen biology students interested in pursuing research positions in college (PSP).<br />
|tech= Price of one 12 oz Styrofoam cup: 4.95 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 16th<br />
|text=The Gibson sequencing came back positive! We then digested pC13X, pC13F, pC13Z and pC13P, and ligated pAK13D to inserts pC13Z, pC13X and pC13F, and pC13P to inserts to inserts pC13Z, pC13X and pC13F. We ran a PCR for pC13M, pC13E and pC13K. Sadly pC13K did not work… Transformation was attempted for pC13AI and pA13AJ but they both failed--we suspected that the cuvettes weren't cleaned properly. <br />
<br />
|tech=Sequencing results came back positive for the Gibson cloning of hph, so a culture of Gibson colony 5 was made. pC13AH was transformed and plated, and pC13K were miniprepped. pC13X (SpeI/PstI), pC13F (SpeI/PstI), pC13Z (SpeI/PstI) were purified and digested along with pC13P (XbaI/PstI) [also dephosphorylated], then all were column purified. Reran a PCR of pC13M and pC13E, and one of pC13K with primers 31/32. All PCRs were cleaned, then run on a gel along with pBARGPE1 which had been digested with NotI today. Results: pC13E had a 3kb fragment (good), pC13K had a .9kb fragment (should be 1.7-1.8kb), pC13M had a 1.2-1.5kb fragment (good), pBARGPE1 had a 4kb fragment (super-coiled) and pBARGPE1 with NotI had a 5-6kb fragment (has a NotI site-good). From these results it can be concluded that everything worked except pC13K. Ligations of pAK13D to inserts pC13Z, pC13X, and pC13F along with vector pC13P to inserts pC13Z, pC13X, and pC13F were performed. Also, overnight digestions of pC13M, pC13E, and pC13K with XbaI and PstI were set up. Transformation was attempted for pC13AI and pA13AJ, but the cuvettes weren't cleaned properly so it failed. Finally, our dwindling supplies prompted the ordering of more SpeI and membrane filters. <br />
|author=Danielle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 17th<br />
|text=We reran a PCR for pC13K and the band was as expected on the gel! pC13M was ligated to pC13D, and pC13E was ligated to pC13P. The resulting constructs were used to transform ''E. Coli'' cultures. The following transformations were also done: ''pT7'' + ''GFP'', PtrpC + ''GFP'', ''pT7'' + ''BAR'', PtrpC + ''BAR''. A colony PCR was run for pC13AH.<br />
<br />
|tech= A glycerol stock of pCh13V (''hph'' Gibson) was created, then miniprepped for later site directed mutagenesis of ''hph'' (which has both E and P cut sites). Digestions of pC13M (crtB) and pC13E (T7 polymerase) were column purified and quantified. The PCR of pC13K with GC and non-GC trials was redone, since the fragment was of the incorrect length during gel imaging yesterday - goal is to ligate to pAK13D. Following the PCR, a gel was run which showed no bands. Perhaps the polymerase was forgotten? Another PCR of pC13K was performed and run on gel. A band showed at exactly 1.8kb as expected. pC13M was ligated to pAK13D, placing crtB downstream of the T7 promoter. pC13E was ligated to pC13P, placing the ''T7'' polymerase gene downstream of PtrpC (a native fungal promoter) - this is the first part of our genetic circuit with the second being the ''T7'' promoter upstream of various genes (GFP and RFP for diagnostic purposes - to quantify promoter functionality and the carotenoid genes for proof of concept). Plates of pC13AH (T7 + crtY) had many colonies, so they were restreaked, and colony PCRs were run while cultures were made of some of the colonies. A gel of the PCRs was run (image name was pC13AHcolonypcr7-17-13); gel was inconclusive (correct band was accompanied by strong incorrect band at 1kb). Will test again using digest screen of minipreps tomorrow. Heat shock transformed ''pT7'' + ''GFP'' (pAK13D + pC13Z), PtrpC + ''GFP'' (pC13P + pC13Z), ''pT7'' + ''BAR'' (pAK13D + pCp13X), PtrpC + ''BAR'' (pC13P + pCp13X) ligations into chemically competent E. coli. Heat shock transformed pC13AI (lox site), pA13AJ (lox site), pC13AK (mRFP coding sequence), and pK13AL (mRFP coding sequence) from Kit Plates 3 and 5 into chemically competent E. coli. Heat shock transformed today's earlier ligations: pAK13D + pC13M, and pC13P + pC13E and plated all transformations in accordance with the selection marker on the plasmid.<br />
|author=Danielle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 18th<br />
|text=The restriction cocktail method of cloning worked! Cloning of pC13AH (''T7'' + ''crtY'') was shown to work using this method. We are also continuing our cloning of the ''crtI'' and ''crtB'' pathways. <br />
<br />
|tech=All 18 (yes, 18!) of the ''pC13AH'' colonies that had been screened by colony PCR yesterday were miniprepped. We digested the miniprepped colonies of pC13AH clones (T7 promoter with crtY in pSB1C3) with ''NotI'' to see if the restriction cocktail method of cloning worked. On the gel that was subsequently run, bands were expected at ~2.1 for pSB1C3 and ~1.2 for ''crtY''+''pT7''. The restriction cocktail method worked! The bands appeared at the correct lengths on the gel for ''pC13AH''. There was not enough miniprepped DNA for pC13AH to send for sequencing, so the colonies were cultured again for another miniprep. None of the ligations into pAK13D (pSB1AK8 + PT7) worked -- pC13Z (''GFP''), pC13M (''crtB''), and pCp13X (''bar'') all failed => try cloning only using restriction cocktail method, instead of attempting PCR with VF2 & VR. Digested pC13Z, pC13M, pCp13X, pC13K (''crtI'') with XbaI and PstI in addition to ApaLI and SacI for later ligation with pAK13. pAK13D had been previously digested with SpeI and PstI, column purified, and dephosphorylated for insert ligation. More pAK13D was grown to acquire more DNA to be miniprepped tomorrow. Nupur performed colony PCR and made cultures of transformants from yesterday. All transformations appeared to have very low efficiency--perhaps an issue with heat shock stocks? Transformations of ''AI-AL'' (mRFP cds and lox site parts) all yielded colonies, though at really low efficiencies compared to most transformations off of kit plates. These were screened by colony PCR to verify. Ligations into pC13P all yielded colonies, but at very low efficiencies. These were pC13E (T7 pol), pCp13X (bar), and pC13Z. All were screened by colony PCR as well. Taq was used to perform 5 colony PCRs, then all colony PCRs were run on a gel. Also, pCg13S (nptIIBB)was digested with XbaI, PstI, ApaLI, and SacI for later ligation to pC13P, which had been previously digested with SpeI and PstI, column purified, and dephosphorylated. Overnight ligations of pC13Z, pC13M, pCp13X, pC13K, pCg13S with vector pAK13D were set up, along with overnight ligations of pC13K and pCg13S in vector pC13P. <br />
|author=Danielle<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 19th<br />
|text=Sent pC13AH in for sequencing, and checked several plasmids for the presence of the correct genes using digestion screens and gel analysis.<br />
|tech=Made glycerol stocks of pC13AT, pC13AY, pC13AV, pC13AI, pA13AJ, pC13AK, pK13AL, and pAK13D. Miniprepped pC13AH cultures and sent in for sequencing. Ran a digest screen on pC13AI, pA13AJ, and pC13AL using NotI. Reran a PCR of pC13AV and pC13AT with Q5 polymerase. Attempted transformation of yesterdays ligations, with success only on pAK13AN and pAK13AD. A gel was run of pC13AL, pC13AJ, pC13AI (all digested w/ NotI), as well as the PCR of pC13AV and pC13AT. Plasmid pC13AT appeared to have worked, as well as pC13AI and pC13AJ, which had strong bands in the ~2kb range and weak bands in the ~1.5kb range, about the size of the whole plasmid.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 20th<br />
|text=Plasmid pC13AY was prepared for sequencing, and several transformations using heat shock were performed.<br />
|tech=Plasmid pC13AY was prepared for sequencing. Transformed pC13BD, pCg13AY, pAK13AC, pAK13BB, pAK13AB using heat shock. Recovered for ~1hr 10min, then plated on ampicillin plates made by spreading antibiotic on a sterile plate. More ampicillin plates were then created from scratch.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 21st<br />
|text=Made cultures of the previous days transformants, and ran an inconclusive digest screen of pAK13AN and pAK13AD. A slow day.<br />
|tech=Miniprepped pAK13AN, pAK13AD and ran a digest screen using NotI. The gel was inconclusive. The previous days transformants (pC13BD, pCg13AY, pAK13AC, pAK13BB, and pAK13AB) were cultured.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 21st<br />
|text=This week, the fabrication group focused on what materials to construct our chamber from. At first we considered a single wall design, but this was eventually scrapped, as we were unable to find any material that would provide sufficient insulation at a reasonable thickness (and cost). After putting our heads together, we decided on a double wall with an insulating layer in between them. Mac began drawing up the chamber in Solidworks. <br />
|author=Mac <br />
}}<br />
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<div id="week6entry" class="nb-week""><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 6</h3><br />
<h5 class="centered">(07/22 - 07/28)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 22nd<br />
|text= Sent in pC13AH, pC13AY, and pC13AT for sequencing. In addition, the digest screens of all the pAK13D ligations were redone, along with the colony PCRs of the pC13P ligations. Transformed pC13AV. Attempted to dry mycelium to isolate genomic DNA.<br />
|tech= Cultures pAK13AB number 5, pCg13Y number 1, pAK13AC number 1, and pCg13Y number 3 from yesterday didn't grow for some reason. Ran a NotI digest screen of all the pAK13D ligations, as well as colony PCRs of the pC13P ligations. Plasmid pAK13AB numbers 1, 3, 4 and possibly pAK13BB number 4 looked like they worked on the gel. The gel of the colony PCRs looked weird, possibly because they were run on wrong extension time for a while. Going to rerun PCRs. We also re-ran a gel of the NotI digest screens from yesterday, pAK13AP and pAK13AN. All the pAK13AP colonies had blank colonies, but pAK13AN colony 1 had the correct bands. Sent pAK13AN colony 1 for sequencing but accidentally sent in the wrong primers. Primers for pSb1AK8 should have been used, but pSB13C primers were used instead. Transformed pC13AV ligation and pC13AL. The transformation arced, but the time constant was still 3.8 so the cells were plated anyway. Finally, 2 250mL solutions of cym agar were made and autoclaved. Began drying mycelium to isolate the genomic DNA.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 23rd<br />
|text= Miniprepped and made a glycerol stock of pAK13AN. Also retried the cloning for pAK13AP and pAK13AC and began cloning for pAK13AO. Plasmid pC13AH was sequence confirmed!<br />
|tech= The plate of pK13AL transformation had no colonies, so it was given more time to grow. The plate of pC13AV had a lawn, but also lots of small isolated colonies. Plate was re-streaked to get fewer colonies before culturing. In addition, miniprepped and made a glycerol stock of pAK13AN and re-ran a colony PCR of pC13BD and pCg13Y with Taq polymerase. Ran a gel to see if ligation worked, but the bands were too short. This may be due to using Taq instead of Q5, so the PCR was run again with Q5. Retried cloning pAK13AP and PAK13AC, digesting pAK13D with SpeI and PstI, and pC13X and pC13K with XbaI and PstI. Overnight ligations were created, after column purification and dephosphorylation. We will redo a digestion of pC13AK tomorrow because we digested with SpeI and PstI instead of XbaI and PstI. We began cloning of pC13AQ, pC13BC and pC13AF, digesting backbone and insert with EcoRI and PstI. We made more DH5alpha electrocompetent cells to try to make heat shock. To replenish, TAE and LB were made. <i>G. lucidum</i> finished drying.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 23rd<br />
|text=The parts have arrived and Rebecca configured them. Met up with Paras who was on last year's team to get a detailed explanation of how their project had worked as well.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 23rd<br />
|text=Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 24th<br />
|text= Ran a gel of the Q5 PCR of the pCg13Y and pC13BD. One sample of pC13BD looked good, so it was sent in for sequencing. Isolating <i>G. lucidum</i>'s DNA didn't work. Will try flash freezing the cells with liquid nitrogen next time. <br />
<br />
|tech= Ran a gel of yesterday's Q5 PCR of pCg13Y and pC13BD, but only one sample of the latter looked good and was sent in for sequencing. Isolating <i>G. lucidum</i> DNA did not work because it did not precipitate. Next time, we will freeze the cells with liquid nitrogen. Re-tried cloning of pC13AY, pC13AT, and pCg13Y. Plasmids pC13E, pC13Z and pCg13S were digested with XpaI, PstI, SacI, and ApaII, while a previous digest of pC13P was used. Subsequently, pC13E, pC13Z, and pCg13S were column purified and the plasmids left to ligate overnight. Tried to redo cloning of pC13AU and pAK13AO because pC13AK was digested with the wrong enzymes yesterday, but failed once again. Finally, pC13I was cloned and pC13BD and pC13Z were submitted for sequencing.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 25th<br />
|text=We tried the site directed mutagenesis of pCh13V to remove the Pst1 internal cut site. Looking at the gel, it looks like the site directed mutagenesis worked! In addition, we cloned <i>crtE</i> into pSB1C3 and performed a ton of transformations!<br />
<br />
|tech= After growing up more pC13AK, attempted site directed mutagenesis to get rid of the PstI internal cut site in pCh13V. After running a gel against an unmutated control, found that GC worked while non-GC didn't. Afterwards, both were digested with DpnI and column purified. Attempted to clone pC13I. Also transformed ALL of the constructs, including: pC13AK, pK13AL, pA13AM, pCp13Y + pAK13AD, pAK13AC, pAK13AO, pCg13Y, pC13AY, pC13AU, pC13AV, pC13AT, pC13AQ, pC13AF, pC13I(crtE in psB1C3), and pCH13V GC with PstI mutation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 26th<br />
|text= Sadly, all 20 transformants failed and the stocks are incompetent. We received new cuvettes to retry all of them. We submitted ''lox'' site for sequencing of pBARGPE1 and ran a PCR of pC13F to append Kpn1 and BamHI restriction sites to pSB1C3 for cloning of “PpelA”.<br />
<br />
|tech= Sequencing results showed that pC13BD had the correct reverse sequencing, but the forward sequencing is being re-run. In addition, all 20 transformations failed due to incompetent stocks. Miniprepped and made glycerol stocks of pC13AK for more DNA and made many new plates. Also submitted the <i>lox</i> site for sequencing and ran a PCR of pC13F to append Kpn1 and BamH1 restriction sites to PSB1C3 for cloning of <i>PpelA</i>. We also ran PCRs using primers 27 and 28 to amplify <i>A. nidulans</i> <i>PgpdA</i> out of pAb13T, and ran a PCR using primers 29 and 33 to amplify <i>crtE</i> + <i>rbs</i> for pC13J out of pC13A. When all was said and done, the gel showed that only the PCR of <i>A. nidulans</i> looked promising, so we cloned this into pC13F.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 27th<br />
|text= We retried the PCR of pC13F, ran 63 colony PCRs, and made 97 cultures!<br />
|tech= Prepped pC13BD for sequencing, and retried the PCR of pC13F to append KpnI and BamHI restriction sites to clone <i>PpelA</i>. Ran 63 colony PCRs and made 97 cultures of all the previous ligations/transformations from two days ago. Also ran gels of the colony PCRs, but forgot to run the pC13F ones.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 28th<br />
|text= We digested pC13F and miniprepped cultures of yesterday's successful colony PCRs. <br />
|tech= Ran PCRs of pC13F on a gel to check for <i>PpelA</i>. The Non-GC PCR products looked good, but the products in which we used the GC Enhancer did not work. Cleaned up the non-GC enhancer PCR of pC13F and digested it with Kpn1 and BamH1. Also digested pHNU with Kpn1, BamH1, and Sac1. Then the double digest products were ligated overnight. The cultures of pA13AM (they did not grow before) were also retried. Miniprepped the cultures from yesterday whose colony PCR gel screens looked promising. Prepped the colony PCRs for: pC13AQ number 1, pC13AV number 5, pC13V PstI mutant numbers 2 and 3, pC13AF 4.3 and 4.5, and pCg13Y number 1. Also retried the questionable colony PCRs with Q5 off of the minipreps: pC13AY numbers 1-3, pC13AU numbers 1 and 5, pC13I numbers 4 and 5, pC13AT numbers 2 and 3, pAK13BC numbers 1 and 5, and pC13AF numbers 3.2 and 3.3. We digest screened all non-chloramphenicol minipreps using NotI-HF for pAK13AC numbers 1-5, and pAK13AO from July 23rd numbers 1-5. For everything else EcoRI and PstI was used.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 28th<br />
|text=This week, the fabrication group finalized the incubator design and ordered the construction materials! We decided to construct the outer and inner layers out of HDPE plastic in the rather lab appropriate color of white. For our insulation, we decided to use high density polystyrene. After comparing the K-factors for various types of insulation, we determined that this material gave us the most bang for our buck, so to speak. <br />
|author=Mac <br />
}}<br />
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<div id="week7entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 7</h3><br />
<h5 class="centered">(07/29 - 08/04)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 29th<br />
|text=Reran many PCRs and other procedures.<br />
|tech=Ran a gel of the questionable PCRs from yesterday. Plasmids pC13AY number 1, pC13AU number 5, pC13I number 5, pAK13BC number 5, and pC13AF number 3.2 looked good, but the gel was smeary so will re-run tomorrow. From the digest screens, pK13AL numbers 1-3, pAK13AP numbers 1-3 and 5, and pAK13AC number 1 looked good on a gel. The site-directed mutagenesis of pCh13V was continued, with a PCR of pCh13V run to mutate the EcoRI site. Sample number 2 of the non-GC samples appeared to have worked when run on a gel, so a DpnI digest and transformation were performed. Miniprepped pA13AM and ran a colony PCR, revealing that it was not in pSB1C3 as expected and thus was thrown out. Ran a digest screen of pA13AM with NotI to be checked on a gel tomorrow. <i>PpelA</i> ligated into pSB1C3 and transformed. Finally, after a long day, retried the PCR of pC13A to obtain <i>crtE</i>.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 30th<br />
|text=Yesterdays transformations all failed, and other mishaps. But fungal DNA extraction was successful!<br />
|tech=Submitted pC13BD, pC13AT, pC13AV, pCh13V mutant and pAK13AB for forward sequencing. Yesterdays transformations failed. A digest screen of pC13A and pC13AM revealed that the primers for pC13A were not functioning, and still no pC13J. Reran colony PCRs from gel yesterday. Digested pC13AL with XbaI, PstI-HF, ClaI, and SacI-HF and ligated into pAK13D, as well as digested pAK13AP and pAK13AC with XbaI and PstI-HF and ligated both into pSB1C3. Re-ran the ligation of <i>PpelA</i> into pSB1C3, as well as the mutagenesis PCR of pCh13V. Fungal DNA was successfully extracted!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 31st<br />
|text=Successful returns from sequencing of pC13BD, pCg13Y, pAK13AN and pC13V PstI mutant. In other news, the PCRs of <i>Ganoderma</i> DNA looked good!!<br />
|tech=Column purified pC13W DpnI digests: 3 GC and 3 nGC transformed and plated. Dephosphorylated pA13AM and ligated with pC13E. Transformed ligations from yesterday: pAK13AO, pC13AS number 3, pC13AG number 1, and pC13BF. Successful returns from sequencing of pC13BD, pCg13Y, pAK13AN and pC13V PstI mutant. Transformed and plated <i>Aspergillus</i> <i>Pgpd</i> in pC13F GC and non-GC.<br />
<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 31st<br />
|text=Sara and Rebecca met up with our advisor Dr Bruce Land and he provided lots of helpful advice with regard to the design considerations (where the heater should be in our incubation chamber, convection currents, type of heating plate to use). He also suggested that we use a proportional-integral-derivative controller (PID controller) to regulate the heating.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 1st<br />
|text= Ligation of the ''PpelA promoter'', hygromycin resistance, and ''T7 promoter'' in front of the herbicide-resistance bar gene worked. We continued cloning of our other constructs and repeated PCRs of <i>G. lucidum</i> GPD homology regions.<br />
|tech= We did colony PCRs with biobrick primers 31 and 32. We also made cultures of yesterday's transformations: pC13D+pK13AL, pC13D+pCp13X #3 in pSB1C3, pC13D+pC13K #1 in pSB1C3, pC13W with GC #3, pC13W no GC #3, and ''PpelA'' in pSB1C3. A gel was run to see if the ligations were successful. pC13D+pC13X, PpelA, pC13W GC 3.1 and 3.5, and pC13W nGC 3.1 all looked like they worked. The pA13AM+pC13E ligation from yesterday was transformed and plated. <i>G. lucidum</i> GPD homology regions were cloned, column purified, and digested with EcoRI and Pst. pC13F was also digested with EcoRI and Pst. Unfortunately, digestions of the homology regions yielded no DNA, but digestion of pC13F did yield some and thus was dephosphorylated. There was not enough template from the PCR cleanup present for homology regions, so PCRs were redone for each homology region.<br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 1st<br />
|text= Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 2nd<br />
|text= We continued working with the PCRs of the GPD homology regions and the successful ligations of the previous day's PpelA promoter, hygromycin resistance, and T7 promoter in front of the herbicide-resistance bar gene, as well as T7 polymerase behind a constitutive promoter and <i>Aspergillus</i> promoter. <br />
|tech= We cloned <i>G. Lucidum</i>GPD homology regions (pC13BJ, pc13BK, pC13BL, pC13BM). Yesterday's PCR reruns were run on a gel to make sure the right regions were amplified. All have correct bands! We column purified the reruns and digested with EcoRI and PstI. We also digested more ''pC13F'' with EcoRI and PstI and dephosphorylated the vector. We purified and ligated the reruns with pC13F. The cultures that looked good from yesterday's colony PCRs were miniprepped. We then made cultures and did a colony PCR of A.Pgpd in pSB1C3 GC from July 31st. A gel of the colony PCRs was run - colonies 1, 3, 4, and 5 looked successful and had the correct band insert length. A transformation of pA13AM+pC13E ligation did not work since the plate was contaminated. Sequencing of pC13AS #1, 3, 5 and pCh13W (hph BB) GC 3.1, 3.5, nGC 3.1 was prepared. An overnight culture of PpelA #2,3,5 was set up for miniprepping tomorrow and sequencing. We transformed pC13P+pC13E #3 and plated for cultures tomorrow, which we will then miniprep and sequence.<br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 2nd<br />
|text= Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 3rd<br />
|text= We continued cloning of T7 polymerase behind a constitutive promoter, transforming the GPD homology regions, and biobricking the carotenoid and geneticin resistance genes. <br />
|tech= We inoculated five colonies of pC13AY and five colonies of pA13AM+pC13E. We performed colony PCR of pC13AY transformants. A gel was run, but we realized that the PCR failed due to the use of Taq instead of Q5 and a 1 min shorter cycling time than the protocol required. We grew a culture of pC13P for miniprepping, digesting with Spe and PstI, and ligation with pC13Z and pK13AL. A digest screen of pAK13AO minipreps was performed - they were incorrect. pC13M, pC13K, pC13Z, pK13AL, and pCg13S were digested with Xba, Pst, Sac, ApaLI. An overnight ligation of pC13M + pAK13D , pCg13S + pAK13D, pC13K + pAK13D was performed. PpelA and A.Pgpd cultures were miniprepped. pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BM, and pC13F+pC13BL were all electroporated and plated. <br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 4th<br />
|text= We grew cultures of the GPD homology region transformations and transformed yesterday's carotenoid genes and geneticin resistance constructs.<br />
|tech= We miniprepped pC13AY, pC13AM+pC13E colonies, and pC13P. The minipreps were stored in the fridge, and glycerol stocks were made in case one of them works. We grew overnight cultures of five colonies from pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BM, and pC13F+pC13BL from yesterday's transformations. <br />
We transformed and plated pC13M + pAK13D, pCg13S +pAK13D, and pC13K +pAK13D. More LB agar and ampicillin plates were also made today.<br />
|author=Tina<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 4th<br />
|text=Mac went to visit Ecovative Design with Swati, Rafael, Alex, and Hannah. We were able to meet with Gavin McIntyre, one of the founders of the company, who was kind enough to give us a tour of their facilities. He also gave our wetlab members some pointers for our own project. In other news, we are still waiting for our materials to arrive. <br />
|author=Mac <br />
}} <br />
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<div id="week8entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 8</h3><br />
<h5 class="centered">(08/05 - 08/11)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 5th<br />
|text= Our previous construction of pC13AY failed. We submitted pC13BF and pC13BG for sequencing. We ran a colony PCR for pAK13AB, pAK13AC and pAK13BB. A few pAK13AC and pAK13BB colonies were successful. <br />
<br />
|tech= We electroporated pA13AM into ''DH5α'' and plated on ampicillin infused plates. We will know if the transformation was successful by the presence of red colonies as a result of ''mRFP'' in the plasmid. We found that our construction of pC13AY failed after running a digest screen as none of the lanes displayed bands at around 3kb, which corresponds to inserted ''PtrpC'' and ''T7 polymerase'' and 2kb corresponding to the vector. However, the gel may have been showing unique banding patterns for the construct, so another gel will be run to check for any supercoiling or linearized plasmids. We also submitted our constructs pC13BF and pC13BG for sequencing. Colony PCRs were done for pAK13AB, pAK13AC, and pAK13BB. A few pAK13AC and pAK13BB colonies were successful. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 5th<br />
|text= Eric Holmes and Arun Chakravorty presented for a group of scientific researchers (SILS)<br />
|author=Hannah<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 6th<br />
|text= After the previous failure of pC13AY construction, we recreated it with successfully screened parts. We redid a colony PCR for pAK13AB, which failed again. We will need to reconstruct pAK13AB. <br />
<br />
|tech= Yesterday’s transformation of pA13AM yielded no transformants, so we retried the transformation today. We ran a digest screen of ''PtrpC'' and ''T7 polymerase'' parts and found that the ''PtrpC'' used for our previous construction of pC13AY was of poor quality. We then set up another ligation of ''PtrpC'' and ''T7 polymerase'' using successfully screened parts. For fluorescence characterization, we double digested pAK13D and pC13P with SpeI and PstI for subsequent ligation with ''GFP'' and ''mRFP'' inserts. We then quantified our construct containing the <i>G. lucidum</i> homologous regions in pSB1C3, but we had a low yield. We will wait for a new miniprep kit to grow new cultures and perform the miniprep again. We redid a colony PCR for pAK13AB for extra colonies. They all failed so we need to reconstruct pAK13AB.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 7th<br />
|text= We received sequencing back for pC13BF and pC13BG and only the later was successful, so we restarted construction for pC13BF. We also ran a PCR to amplify ''GFP'' and ''mRFP'' inserts out of their respective plasmids.<br />
<br />
|tech= We designed primers for ''lox'' Gibson assembly and appended cut sites to genes flanked by ''lox''. We also found a possible viable kill switch in the Holin/Chitinase system. Our second try at transforming pA13AM was successful as shown by the presence of red colonies and we set up LB cultures for subsequent cloning. Sequencing showed that construction of pC13BG was successful and cultures were made for further use. However, sequencing of pC13BF was unsuccessful and we reran the PCR of pC13F in order to append BamHI and KpnI cut sites for ligation with pA13BE. We ran another digest screen for pC13AY but many lanes did not have bands so we will retry it tomorrow. We also amplified ''GFP'' from pC13Z and ''mRFP'' from pC13AK using primers 23 and 24 in a Q5 PCR. Afterwards, a gel screen on PCR products was run to verify that the correct genes were amplified. The gel image indicated consistent bands with lengths of 0.7 kb, thus the PCR to obtain ''GFP'' and ''mRFP'' inserts was successful! We made a rough draft of our protoplasting protocol to attempt in the near future, too. We then rummaged through previous notebook entries to figure out the problems with ''crtB'' and ''crtI''. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=August 7th<br />
|text=We helped [[Team:Ciencias-UNAM|Ciencias-UNAM]] get set to transfer their HTML website to the wiki. Hopefully we'll get up a tutorial for what to look out for on here soon!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 7th<br />
|text=The sensor has been connected! <br />
|author=Rebecca<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 8th<br />
|text= Cloning of the ''Cre'' gene and ''PpelA'' is going well, and we submitted ''PT7'' with ''GFP'' for sequencing, as well as pC13AS, pC13W, pAK13BB, and pAK13AC. <br />
<br />
|tech= We ran a PCR of pA13BW. The ''CRE'' gene amplified correctly and the sample was column purified, yielding 54.8ng/uL (with GC enhancer) and 49.9ng/uL (with non GC enhancer). We ran out of supplies to miniprep, so we pelleted pA13AM and pC13BG cultures and began to not so patiently await the arrival of a new miniprep kit. Our PCR of pC13F to append BamHI and KpnI cut sites yesterday failed completely, so we digested pC13F with KpnI to linearize it with an internal cut site in ''mRFP'' for another PCR to be run tomorrow. Another gel was run of the pC13AY digests and all lanes were blank. There may have been an issue in quantification by nanodrop that was reading over 1000 ng/μL that mislead us into using low volumes of DNA for digestions. We decided to electroporate with the remaining pC13AY to miniprep and redo gel screening. On a brighter note, our restriction cocktail digestion of pA13BE with BamHI, KpnI and SacI showed a band at 500bp, which is the approximate length of ''PpelA''. We also searched for a pAK13AN miniprep in the DNA storage box and for a glycerol stock of the same plasmid. We couldn’t find either. However, we did obtain a plate of colonies that had been transformed with pAK13AN. We picked colonies off this plate to miniprep and send off to sequencing. In addition, we column purified our inserts from the PCR run yesterday of pC13Z and pK13AL. We then quantified inserts after column purification. After, we digested backbones pAK13D and pC13P with SpeI and PstI and vector dephosphorylated both. The inserts, ''GFP'' in pC13Z and ''mRFP'' in pK13AL were then digested with XbaI and PstI. We ran a gel of backbones and inserts for confirmation of digests. Unfortunately, the results showed no bands from DNA. Perhaps there was not enough DNA from backbones for proper visualization. We also received materials for protoplasting today, and autoclaved or sterilized the supplies in whatever way we could. We made a filtration system such that all the filtering process during <i>G. lucidum</i> protoplasting will remain sterile. We made another stock of <i>G. lucidum</i> in CYM media, as well as 25 mL cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM. We made glycerol stocks of pC13M and pC13K from glycerol stocks. We submitted pAK13AC for sequencing and ran a Q5 PCR for amplifying insert for constructing pC13I from pC13A. <br />
|author=Jonlin <br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 9th<br />
|text= Cloning of the Cre gene is going well, and we may have found a useful limonene synthase gene! We are continuing the previously problematic cloning of ''GFP'' with T7 promoter-hopefully it works this time! Sequencing of pAK13AC unfortunately failed. <br />
<br />
|tech= We digested both the PCR products of ''Cre'' and pC13F with EcoRI and SpeI. We may have found a limonene synthase gene we can use. We ran another PCR to append cut sites to the linearized pC13F that ultimately failed with no bands visible on a gel. As our previous construction of pC13AY likely failed since no colonies being present on the new plate, we redid digestions of pC13E and pC13P to redo the construction. We also redigested both pAK13D and pC13P minipreps with SpeI and PstI since the gel from yesterday was inconclusive. We then ligated pAK13D with ''GFP'', pAK13D with ''mRFP'', pC13P with ''GFP'' and pC13P with ''mRFP''. We prepared pellets from our cultures of pC13F+pC13BK, pC13BL, pC13BJ, and pC13BM cells which were stored in the fridge as we wait for our miniprep kit to arrive. Sequencing of pAK13AC has failed, and we will resubmit a miniprep of it. We plated stocks of pC13M and pC13K. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 10th<br />
|text= We began constructing pC13BH with Gibson assembly, as well as making our first attempt at protoplasting <i>G. lucidum</i>. Electroporations of fluorescence constructs was also done today. <br />
|tech= We did a fast ligation (30 minutes, room temperature) of ''Cre'' with pC13F and electroporated. The time constant was low, so the transformation may not have worked. We continued the ligation overnight. We were still waiting on a miniprep kit so we pelleted our pA13AM and pC13BG cultures to prevent further growth. We transformed with our new pC13AY ligation to hopefully get some colonies this time. We also ran a PCR of pC13F to append homologous regions to <i>G. lucidum</i> ''PgpdA'' for Gibson assembly to create pC13BH and another PCR to retry appending BamHI and KpnI cut sites to pC13F using half to reaction volume to see if efficiency would be improved. We also electroporated the 4 ligations for fluorescence from yesterday (pAK13D with ''GFP'', pAK13D with ''mRFP'', pC13P with ''GFP'' and pC13P with ''mRFP'') along with a control of pC13F to make sure that the electrocompetent cell stocks made yesterday are good. We plated the electroporated cells onto selective antibiotic resistance plates and incubated in 37 degree incubator overnight. We made our first attempt at protoplasting <i>G. lucidum</i> today. However we were having difficulty forming pellets of protoplasts, so we viewed the samples under a microscope. It appeared that the protoplasting attempt was unsuccessful. pC13M and pC13K plates were lawns on their plates. We set up overnight cultures of pC13AS, pC13W, and pC13Y, planning to miniprep and make glycerol stocks.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 10th<br />
|text= Five team members presented at the Ithaca Sustainability Center for citizens of Tompkins County.<br />
|author=Hannah<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 11th<br />
|text= We are starting cloning of limonene synthase and continuing cloning of the ''Cre'' gene. <br />
Cloning of fluorescent constructs is going well. <br />
|tech= The electroporation from the 10th did not work (there were no colonies). Electroporation was redone on the overnight ligation of ''Cre'' and pC13F and a better time constant was observed (even with poor cuvette drying). We transformed limonene synthase from 2013 kit plate 4. We discovered that we need spectinomycin to use the holin parts registry plates. We got colonies from the pC13AY transformation and cultured them for gel screening. For fluorescence, no colonies were found on pAK13AN and pAK13AO plates. We centrifuged potential pC13AQ cultures for 10 min at 5000g and poured off supernatant. We kept the cell pellets in the fridge for mini prepping tomorrow. We also found two colonies each on the PtrpC and ''GFP'' plate and PtrpC and ''mRFP'' plate. We picked off all the colonies and grew each up in 5 ml liquid LB with CM antibiotic. pC13M and pC13K plates are now ready for miniprepping. We could not miniprep the cultures of pC13AS, pC13W, and pC13Y due to running out of our miniprepping supplies.<br />
|author=Rafael<br />
}}<br />
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<div id="week9entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 9</h3><br />
<h5 class="centered">(08/12 - 08/18)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 12th<br />
|text= Cloning of PtrpC and ''GFP'' appears to be going well. Our initial attempt at protoplasting and transforming <i>G. lucidum</i> does not appear to have been successful.<br />
|tech= We still didn’t receive a miniprep kit so we pelleted the pC13AY cultures. The PCRs we ran on pC13F to append homologous regions and append cut sites may have been successful. DNA was present but in low concentrations that we could not check on a gel. We had transformants for pC13BX and pC13CJ so we ran a colony PCR and made cultures. The colony PCRs showed little to nothing on the gel, perhaps because Taq polymerase lost activity. We will miniprep the culture and use Q5. We also attempted a restriction cocktail digest today for both ''mRFP'' and ''GFP'' inserts. We cut pC13Z and pC13AK with Xba, PstI, SacI, and ApaLI. We also performed a colony PCR of pC13AT cultures and pC13AU cultures using primer 23 and 24 to verify length of inserts. The lengths should be around .7kb for each insert. When we ran a gel of the PCR products, the colony PCR showed a .7kb insert for pC13AT colony 1 and a .3kb insert for pC13AT colony 2. The bands for pC13AU were inconclusive. We will miniprep the potential pC13AT colony 1 and send for sequencing once the miniprep kit arrives. We digested our plasmid pAh13G with SphI in order to create linear DNA for fungal transformation. We attempted to transform this into what <i>G. lucidum</i> protoplasts we had generated, but the electroporation lead to arcing. Arching most likely resulted from the sodium citrate in the cell solution which we were instructed to use by the literature. Nevertheless, we plated the cells on hygromycin cym agar.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 13th<br />
|text= We continued cloning of ''T7'' with ''mRFP'', PtrpC with ''GFP'', and PtrpC with ''mRFP''. <br />
|tech= We spun down the pC13BX and pC13CJ cultures and put them in the fridge to be miniprepped. We also ligated ''T7'' with ''mRFP'', PtrpC with ''GFP'', and PtrpC with ''mRFP'' since restriction cocktail digests looked good from the gel yesterday. We then electroporated the ligations and plated them. The plates were left overnight in the incubator. We created another culture of <i>G. lucidum</i> this time in an effort to obtain solid pieces at the top of the media instead of mycelium floating throughout. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 14th<br />
|text= Homologous regions were successfully appended to ''PgpdA'', and cloning of the Cre recombinase appeared to be successful. We made another attempt at protoplasting, which was again unsuccessful. For characterization, we began cloning RBS into our constructs containing ''T7'', since the promoter need a ribosomal binding site to express in <i>E. coli</i>. <br />
|tech= We ran a PCR to append homologous regions to <i>G. lucidum</i> ''PgpdA'' for Gibson assembly with pC13F that succeeded as verified by a gel screen, but we accidentally used a RNA binding tube during purification and lost all the DNA. Needless to say, that mistake won’t happen again. Luckily, when we ran the same PCR again it was successful. We ran a colony PCR from the grown cultures of pC13BX and pC13CJ to visualize the insert using Q5. We got a positive insert for pC13BX. So this will be miniprepped and sent for sequencing once the miniprep kit is in. Unfortunately, no colonies appeared on the potential pAK13AO plate. We also decided to transform RBS (pA13CZ) off of a kit plate for subsequent miniprepping/digestion/ligation with ''GFP'' and ''mRFP''. The ''T7 promoter'' needs a ribosomal binding site in order to express in <i>E. coli</i>. We then retried the ligation of pAK13D with ''mRFP'' using a 6:1 insert to vector ratio. We attempted to protoplast a huge chunk of mycelium, but had difficulty forming a pellet again. At this point, we decided to enlist the help of Professor Gillian Turgeon, who performs fungal transformations in her lab. We made some hygromycin plates to test transformants, and digested pAh13G with Sph1 to linearize, but this time we column purified the product.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 15th<br />
|text= We ran a Gibson assembly to create pC13BH and transformed the construct.<br />
<br />
|tech= We ran a Gibson assembly of pC13F and <i>G. lucidum</i> ''PgpdA'' to create pC13BH and then transformed and plated the new construct. As our previous attempts to append BamHI and KpnI cut sites to pC13F failed, we again ran another PCR, but tried using four times the amount of dNTPs. We ran a PCR of pC13CJ once again from the cultures. The gel looks good for colony 4 and okay for colony 3. We will miniprep colony 4 and send it in for sequencing. We also made five 5ml liquid LB cultures of pA13CZ and miniprepped pC13AQ. We ran a digest screen of select pC13F+pC13BJ, pC13F+pC13BK, pC13F+pC13BL, pC13F+pc13BM colonies with EcoRI and SpeI. Our initial gel didn’t show inserts (lengths of 130 and 230 base pairs) so we ran a second gel with 2% agarose. We still couldn’t see inserts, so we ran digest screens on all colonies (1-5) and ran a new gel. We saw very faint bands for some of them, and nothing for others. Additionally, Arun created a device to use the shaking power of our vortex to agitate a sample in the waterbath. We made LB cultures of pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13AC and pAK13BB. We set up new overnight cultures of pC13AS, pC13W, and pC13Y.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 15th<br />
|text=Rebecca looked more into the details of the pulse-width modulation and PID controller so that the heater can be turned on for certain periods of time for more even heating. <br />
|author=Rebecca<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 16th<br />
|text= The miniprep kit finally arrived so we were able to continue progress on our pC13BG, pC13AY and pC13AW constructs. To aid in our protoplasting, we decided to seek help from the Turgeon Lab.<br />
<br />
|tech= The miniprep kit arrived, so we finally were able to miniprep our pelleted pC13BG, pC13AY and pA13AM cultures. We got transformants for the Gibson assembly of pC13BH and we cultured them. The PCR to append cut sites to pC13F again failed, so we need to extensively troubleshoot this PCR. We made large cultures of pC13BX colony 1 and pC13CJ colony 4 to miniprep and sequence. We made electrocompetent stocks of BL21. The symbol is a purple star. We later met with Bradford Condon, a graduate student who works with Professor Turgeon. He gave us a few tips on our method, including using young mycelia, and avoiding the hydrophobic disc that forms on the top of our cultures for protoplasting. The advice was that we should blend <i>G. lucidum</i> which has been growing for an extended period of time, and make new cultures for a short period of time with the fungal "smoothie". We also miniprepped pC13BM, pC13BJ, pC13BL, and pC13BK 6 & 7. The LB stock of pAK13AC was made with the wrong antibiotic so we remade it. We also remade LB stock of pAK13AB. We miniprepped pC13A, pC13D, pC13F, pC13K, pC13L, pC13M, pC13N, pAK13BB, pC13AS, pC13W, and pC13Y. We plated pC13A, pC13D, pC13F, pC13K, pC13L.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 17th<br />
|text= PtrpC with ''GFP'' will be sent in for sequencing. We ran PCR of pAK13AC, pAK13BB, pAK13A (for both ''rbs'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but pAK13A both failed. We ran PCR of pAK13AC, pAK13BB, pAK13A (for both ''rbs'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but both pAK13A failed.<br />
<br />
|tech=Today, we made glycerol stocks of pC13AT and prepared them for sequencing. We then miniprepped pAK13AC. However, the miniprep qualities are poor so we will redo with a larger stock of cells. We ran a PCR of pAK13AC, pAK13BB, pAK13A (for both ''RBS'' + ''crtE'' and ''crtE''). pAK13AC and pAK13BB worked but pAK13A failed. Thus, we retried PCR of pAK13A inserts from a fresh batch of minipreps. We prepared cultures of pC13S, pC13P, pC13Q, and pC13BG.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 18th<br />
|text= Cloning of ''T7 promoter'' with ''mRFP'' has been problematic - but we will keep trying. Minipreps of large stocks of pAK13AB, pAK13AC and pAK13BB yielded better results! <br />
<br />
|tech= Today we miniprepped pC13AT along with pA13CZ. We also ran a gel of PCR products of pAK13AO (5 colonies) in pSB1C3. Insert lengths should be around .7kb, but no bands showed up. We will rerun a gel using more DNA from the same PCR. We ran digest screens of pC13BM, pC13BJ, pC13BL, and pC13BK minipreps 6 & 7, which appear to have all worked. We miniprepped pAK13AB, pAK13AC, pAK13BB, pC13A and plasmid concentration was much higher this time around. We miniprepped pC13S, pC13P, pC13Q, and pC13BG, to prepare for cloning.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=August 18<br />
|text=Today, the dastardly trio of Arun, Hannah, and Eric were chosen to present at the regional iGEM competition!<br />
|tech=<br />
|author=Swati<br />
}}<br />
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<div id="week10entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 10</h3><br />
<h5 class="centered">(08/19 - 08/25)</h5><br />
</html><br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 19th<br />
|text= We are beginning construction of ''T7 polymerase'' with an ''Anderson promoter'', and prepared PtrpC with ''mRFP'' and PtrpC with ''GFP'' for sequencing. Gel electrophoresis showed that pAK13AB, pAK13AC, pAK13BB and pC13A (both ''rbs'' + ''crtE'' and ''crtE'') are good! <br />
<br />
|tech= We ran a PCR of the pC13BH minipreps to verify the success of the Gibson assembly on a gel. We also digested pC13AY in order to gel screen it and check if it is successful. We began constructing pA13AW by digesting and ligating pA13AM and pC13E. We also reran a gel of a colony PCR of pAK13AO. Next, we prepped pC13AT and pC13AQ for sequencing. We sent in pC13BJ, pC13BK, pC13BL, pC13BM minipreps 6 & 7, pC13AS, pC13W, and pC13Y for sequencing. We ran a gel for the PCR products from Aug. 17 and Aug. 18. Everything is good: pAK13AB, pAK13AC, pAK13BB, pC13A (both ''rbs'' + ''crtE'' and ''crtE''). We ran Q5 PCR of pC13N, pC13L, pC13S, pC13Q, and pC13W for creating inserts. We digested pC13S, pC13Q, and pC13W with X and P, and digested pC13BG, pC13D, pC13Y, and pC13P with S and P. We dephosphorylated and set up the ligations of those inserts and vectors to make pC13BQ, pC13BR, pC13CC, and pC13CD.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 20th<br />
|text= Cloning of PtrpC with ''T7 polymerase'' continues to be problematic. We are continuing cloning of ''PpelA'', last time’s ''T7 polymerase'' with an ''Anderson promoter'', and putting a strong RBS with ''GFP'' and ''mRFP''. We digested pC13I, pC13J, pC13N, pC13L, pAK13AB, pAK13AC, pC13F and pAK13D in preparation for ligating new products for the carotenoid pathways.<br />
<br />
|tech= We transformed and plated the pA13AW ligation from yesterday. We also retried the PCR to append KpnI and BamHI cut sites to pC13F again, using the newly received Q5 mastermix and higher annealing temperatures. We ran a massive gel screen and found that the cut site PCR and Gibson assembly may have been successful, but that the construction pC13AY most likely failed. We then digested pC13F with cut sites appended with BamHI and KpnI for ligation with pC13BE to make pC13BF. We digested pA13CZ with SpeI and PstI, which is to be ligated with ''GFP'' from pC13Z and ''mRFP'' from pC13AK. We ligated the Xpa1, Pst1, Spe1, Apa digested pC13AK and Spe1 and Pst1 digested and dephosphorylated pC13BG to make pC13CG, which we transformed into BL21-A1 cells to check transformation efficiency. We also tranformed into DH5α and plated both for comparison. The sequencing results of pC13BJ 7, pC13BK 6, pC13BL 7, and pC13BM 6 confirmed that we were successful, so we made new cultures to create glycerol stocks from, but the initial cultures were thrown out and the plates were difficult to re-pick from, so we hope that cells made it into the culture and have the correct plasmid. We did a PCR clean-up for pAK13AB, pAK13AC, pC13A, pC13A, pC13N, pC13L. We digested pC13I, pC13J, pC13N, pC13L with XbaI & PstI, pAK13AB & pAK13AC with EcoRI & PstI. We also digested pC13F with XbaI and PstI, also with EcoRI and PstI, as well as pAK13D with XbaI and PstI. We transformed ligations of pC13BQ, pC13BR, pC13CC, and pC13CD into <i>E. coli</i>. We also interpreted sequencing results of pC13AS, pC13W, and pC13Y - the latter two look good, and pC13AS may have an overlap, suggesting a mixed population of plasmid.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 21st<br />
|text= Weird happenings in lab: what we thought was PtrpC with ''GFP'' turns out to be PtrpC with ''mRFP''! Good news: ''PgpdA'' and ''pC13F'' with homologous regions for Gibson are good! We tested the transformation efficiency of our new T7-ready bacteria, BL21-A1 as well. We ligated and electroporated pC13AF, pC13AG, pAK13CR, pAK13CS from other digestions, and plated them overnight. <br />
<br />
|tech= We made cultures of the successfully transformed pA13AW and ran a gel screen of the digests of pC13F and pC13BE and found that they did not have enough DNA to be visible. The <i>G. lucidum</i> ''PgpdA'' and pC13F parts with homologous regions needed for Gibson assembly and found them successful. We then ligated and transformed pC13BF although it sparked during electroporation, so we will most likely have to retransform. A sequence confirmation came back for pC13AQ! pC13AT turned out to be PtrpC and ''mRFP''.Thus, we redigested PtrpC in pC13P and pCg13Y with SpeI and Pst1-HF. pCg13Y will be used to create pC13CB. We also redigested pC13Z, pC13AK, and pC13AQ with Xba, Pst1-HF, ApaI and Sac1-HF in order to get necessary inserts. We threw away the cultures of ''pC13BJ" 7, ''pC13BK" 6, ''pC13BL" 7, and ''pC13BM" 6 from yesterday and didn’t make glycerol stocks because we were not sure if we got the correct colonies. We will transform the sequence confirmed minipreps when more DNA is needed. We attempted site directed mutagenesis of ''ptrpC'' + ''hph'' + ''ttrpC'' in pAh13G. The extension time was a little short for pAh13G so it might not work. We ran gel a gel, which had a band at 100 base pairs, meaning it didn’t work. We will attempt this again tomorrow with a longer extension time. Colonies of BL21-A1 and DH5α with pC13CG grew. BL21-A1 seems to have a higher transformation efficiency.We started cultures and performed a colony PCR from the DH5α plate. We ran a gel of the colony PCR and pC13CG 1, 3, and 4 had the correct band at about 1.8 kb. pC13A (''rbs'' + ''crtE'') and pC13F (XbaI,PstI) did not have enough DNA. As a result, ligations for creating pC13I and pC13J are on hold. We ligated and electroporated pC13AF, pC13AG, pAK13CR, pAK13CS from other digestions, and plated them overnight. We found the the transformations of pC13BR and pC13BQ were too efficient and had to restreak the plates in order to isolate single colonies. We ran colony PCRs of pC13BU, pC13CC, and pC13CD, and at least two copies of each construct seem to be successful!<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 22nd<br />
|text= We did a bunch of ligations and are continuing cloning of ''PgpdA'', ''Anderson promoter'' with ''T7 Polymerase'', ''GFP'', geneticin resistance, and a strong RBS. We ligated pC13I and pC13AF. <br />
<br />
|tech= We redid the Gibson assembly of pC13BH trying 1:2 and 1:6 vector to insert ratios and transformed it along with pC13BF as it sparked when we tried yesterday. We also miniprepped our pA13AW cultures. After, we vector dephosphorylated pA13CZ, pC13P, and pCg13Y for fluorescent characterization constructs. We then ligated ''PgpdA'' with ''GFP'', pC13AQ with pC13Y, pC13P and ''mRFP'', pC13P with ''GFP'', pA13CZ with ''GFP'', and pA13CZ with ''mRFP''. All ligations were then electroporated and incubated overnight. We sent pC13BX and pC13CJ to sequencing. We digested and ligated pC13CJ and pAK13D and electroporated into DH5a cells. We miniprepped and made glycerol stocks of pC13CG 1,3,4 (labeled TEMP). We will make sure to go back and throw out extras and failed attempts after sequencing results return. We sent pC13CG 3 in for sequencing, using the last of our DNA, meaning if we need more it will have to come from the glycerol stock. We performed our second attempt at site directed mutagenesis of pAh13G with a longer extension time. We recreated and digested inserts pC13A (''rbs'' + ''crtE'') and pC13F (XbaI, PstI). We ligated pC13I and pC13J. pC13AF electroporation did not yield any colonies. We made glycerol stocks of pC13AG, pAK13CR, pAK13CS, pAK13BU, pC13CC, and pC13CD and put them at -80 degrees C. We miniprepped the successful pAK13BU, pC13CC, and pC13CD and prepared them for sequencing. We also ran colony PCRs of pC13BR and pC13BQ, and set up overnight cultures of the successful constructs.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 23rd<br />
|text= Cloning of RBS + ''GFP'', RBS + ''mRFP'', ''PT7'' + ''GFP'' + PtrpC + geneticin resistance, PtrpC + ''mRFP'', and PtrpC + ''GFP'' appear to be going well. <br />
<br />
|tech= Today we made liquid cultures of and ran colony PCRs of pA13DC, pA13DB, pC13AU, pC13AT, and pC13CB. pC13CF did not grow any colonies. We then ran a gel of the PCR products. pC13CB colony 5, pC13AT colonies 1-3 and 5, pC13DC colonies 2-5, pC13DB colonies 1-5, and pC13AU colonies 1 and 5 are looked like they had the proper insert lengths from the gel image. Sequencing results came back confirming that pC13CG 3 worked We digested our site directed mutagenesis attempt from yesterday with DpnI and stored it in the fridge. We ran a gel combined with other DNA from other team members, which resulted in a smeared band that was in the correct place. We believe this will be successful, and we will transform our site directed mutagenesis product as soon as desalting membranes arrive. We mini-prepped and plated pC13AG, pAK13CR, pAK13CS. We miniprepped pC13BQ and pC13BR, and submitted pC13CC, pC13CD, pC13BQ, and pC13BR for sequencing. We ran PCRs of the inserts in pAK13BB and pAK13BU to prepare for further cloning.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 24th<br />
|text= Running into some problems with ''PgpdA'' cloning. PCR confirmed that pC13AG and pAK13CR worked! We picked more colonies of pAK13CS for screening. <br />
<br />
|tech= We cultured transformants for our Gibson assembly of pC13BH using 1:2 vector to insert ratios, but got no colonies for the 1:6 ratio Gibson and the pC13BF ligation. We also digested our pA13AW minipreps for gel screening. Our PCR confirmed that pC13AG and pAK13CR worked. We picked more colonies of pAK13CS from electroporation plate to screen more colonies. <br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 25th<br />
|text=The extra colonies of pAK13CS from yesterday failed. We will have to restart pAK13CS. We are also recreating inserts from pAK13AB to make pC13AF. <br />
|tech=The extra colonies for pAK13CS that we made yesterday all failed. We will need to restart pAK13CS. We are recreating insert from pAK13AB for recreating pC13AF. <br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 25th<br />
|text=Christine's back! She started looking at the components for a humidity sensor and placed an order for a mistmaker which would be able to control humidity levels. In the meantime, Sara studied the details of the heating circuit which utilizes a transistor to amplify signals and switches it such that the resistor could be used as the heating element. <br />
|author=Rebecca<br />
}}<br />
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<div id="week11entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 11</h3><br />
<h5 class="centered">(08/26 - 09/01)</h5><br />
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<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 26th<br />
|text=Cloning of pA13AW seems to be successful and will test to see if pC13BH also worked. We ran a PCR and digested pC13L to prepare insert for recreating pAK13CS. We recreated ligation of pC13AF.<br />
|tech= The transformed colonies of pC13BH had the ''PgpdA'' insert amplified via colony PCR and gel screened to check for the correct length. The gel however was blurry, so the cultures were miniprepped for digest screening. Gel screen pA13AW digested with XbaI and PstI. Gel results indicate that the cloning was successful! We then amplified the ''T7 polymerase'' gene for more insert DNA. We ran a PCR and digested pC13L to prepare insert for recreating pAK13CS. We recreated ligation of pC13AF. We looked at sequencing results for pC13BQ, pC13BR, pC13CC, and pC13CD - all looked good, other than some minor differences between our copies of the trpC promoter and terminators and the sequences in the online database.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 26th<br />
|text=Our materials are here and everyone in the fabrication group has returned to Ithaca! This week, Manny and Mac focused their efforts on getting the plastic cut to the proper dimensions. Unfortunately, this proved more to be more difficult than anyone could have imagined. After transporting a rather hefty 48"x48" piece of plastic across campus from Weill Hall to the Emerson Lab machine shop, we were told that we would have to take it elsewhere to have it cut (plastic is bad for blades). Disappointed, but never defeated, we decided to try our luck with another machine shop. Also, carrying plastic around campus isn't much fun, so we held it on the roof of a car instead (not recommended). Fortunately, no traffic violations were received, and the Clark hall machine shop was more than happy to cut up our plastic for us. <br />
|author=Mac <br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 27th<br />
|text=Digest screens indicate that cloning pC13F and the ''T7 polymerase'' PCR worked. We are working on the ''cre'' and ''lox'' constructs. pC13AU, pC13AT, pC13CB, pA13DC, pC13AG, and pA13DB were miniprepped and prepared for sequencing. We prepared insert from pAK13CR for creating pC13CV. <br />
<br />
|tech= We made cultures of pC13BX colony 1 and pC13CJ colony 4 to make glycerol stocks. We ran PCR of ''lox'' sites to amplify by Gibson. We ligated pAK13D with limonene. We preppred ''cre'' for reverse sequencing. We transformed ''holin'' off of kit plate and plated it. Digested pC13BH preps using EcoRI and PstI and screened alongside ''T7 polymerase''. Results indicate that only the ''T7'' PCR worked. The backbone pC13F and pA13AM were digested with XbaI and PstI, retrieving the ''T7 polymerase'' gene from the latter. Gel results for the digestions revealed correct banding patterns only for pC13F. We also miniprepped and prepared for sequencing pC13AU, pC13AT, pC13CB, pA13DC, and pA13DB. We prepared insert from pAK13CR for creating pC13CV. We submitted pC13AG for sequencing. We ran PCRs of pAK13BB and pAK13BU, and digested the inserts with EcoRI and PstI. We also digested pC13CD with SpeI and PstI.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 28th<br />
|text=Sequenced pC13BH and ligated ''T7 polymerase'' into a variety of vectors. ''lox'' 2 worked with Gibson, but ''lox'' 1 did not. We created large batch of vectors pC13F and pC13D. The sequence of pC13AG is confirmed! <br />
<br />
|tech= One colony of pC13BH had a good banding pattern and was submitted for sequencing. Digested ''T7 polymerase'' PCR with XbaI and PstI and pC13F with BamHI and KpnI (sites were appended via PCR). pC13F that was digested with XbaI and PstI as well as with BamHI and KpnI were dephosphorylated for ligation. After confirming ''T7 polymerase'' PCR on a gel, it was ligated into pA13AM and pC13P. Additionally, ''Pc + T7 polymerase'' was ligated into pC13F and ''PpelA'' was ligated into pC13F. We ran those PCR's for the Gibson of pC13BN on a gel. ''lox'' 2 worked, but ''lox'' 1 did not, so I purified ''lox'' 2 and reran the PCR of ''lox'' 1 (it is in the small fridge labeled ''lox'' 1). I also made glycerol stocks of pC13BX and pC13CJ so that we have those in case we need to do more minipreps on them. We also had transformants for pC13CN so I made cultures with spectinomycin for all of them and ran a colony PCR on them. The colony PCR did not work due to incorrect protocol. We do not have the correct primers to do a PCR on this plasmid because its in Bbb format, so we just have to assume the plasmid is present because the cultures are growing in Spec. Tomorrow we need to miniprep those cultures that are in the incubator. We blended <i>G. lucidum</i> and made two 100mL cultures with 75mL cells and 25 mL cells. We will now let these grow for 2 days before attempting protoplasting once more. We created large batches of vectors (pC13F (XbaI,PstI) and (EcoRI-HF,PstI), pC13D (XbaI,PstI)). pC13AG sequence is sequence confirmed! We tried to dephosphorylate, but mistakenly used DNA Ligase on pC13F and pC13Q instead, so we’ll have to redo that tomorrow.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 29th<br />
|text=Transformed our ''T7 polymerase'' ligations. We ligated pAK13CS and pC13CV. We electroporated and plated pC13I, pC13J, pAK13CS and pC13CV -- pC13J unfortunately arced…<br />
<br />
|tech=Today we electroporated ''Pc + T7 polymerase'' in pC13F, ''PpelA'' in pC13F, and ''T7 polymerase'' in pA13AM and pC13P. We prepared pAK13AB and pC13AG for glycerol stock. We ligated pAK13CS and pC13CV. We electroporated and plated pC13I, pC13J, pAK13CS, pC13CV. pC13J arced, so we ran a new Q5 PCR to begin preparation of insert from pC13A. We re-digested pC13Q with XbaI and PstI and pC13F with EcoRI and PstI, and subsequently dephosphorylated pC13CD and pC13F. We set up ligations to create pC13BC, pC13BV, and pC13CE.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 30th<br />
|text=Cultures from yesterday’s ''T7 polymerase'' ligation transformations were made. We transformed the ''lox'' Gibson products. pC13AU and pC13AT were successfully sequenced today. We did a PCR with Phusion polymerase in an attempt to amplify the insert in pC13J. <br />
<br />
|tech= Set up cultures for ''Pc + T7 polymerase'' in pC13F, ''PpelA'' in pC13F, and ''T7 polymerase'' in pA13AM and pC13P and ran a colony PCR for each. Each PCR was visualized on a gel; only ''PpelA'' in pC13F appeared to work. We quantified DNA: ''lox'' 1: 72ng/uL, ''lox'' 2: 34ng/uL, pC13CN 640.9 & 820.9ng/uL. We ran Gibson on the ''lox'' PCRs, transformed them into DH5a cells. We made glycerol stocks of pC13CN. We have pC13CK transformants, ran colony PCR and made cultures. We got reverse sequencing on cre and it looks good. We inoculated a culture of DH5a for electrocompetent stocks. pC13AU and pC13AT was sequence confirmed today. However, for pC13CB, PtrpC and ''nptII'' BB re-ligated back to itself pA13DB colony 1 and pA13DB colony 4, pA13DC colony 3 and pA13DC colony 4 all failed to sequence. We discarded all of our pC13CG glycerol stocks except for the successful pC13CG 3, and met with members of Professor Turgeon's lab. We also blended our 25 mL culture from 2 days prior, and made new 100ml cultures with (75 mL and 25 mL, again.) We prepared pAK13AB and pC13AG for glycerol stock. We made glycerol stock of pAK13AB and pC13AG. We redid a PCR with Phusion polymerase in an attempt to amplify ''rbs'' + ''crtE'' (pC13J). We then made overnight E. Coli cultures of pC13I, pC13AF and pC13CV. We transformed the ligations of pC13BC, pC13BV, and pC13CE into <i>E. coli</i>, but both attempts for pC13CE arced, so we started another ligation for pC13CE.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=August 31st<br />
|text= We are continuing cloning of the carotenoid genes and ''lox'' sites. The pC13BN construct looks good. We double digested pC13Z and pC13AK with Xba, PstI, Sac, and ApaLI since we were running low on insert DNA for ''GFP'' and ''mRFP''. We began a new attempt at protoplasting, now with the help of members of the Turgeon Lab. We digested the following miniprep products: pC13I, pC13AF and pC13CV. We also electroporated pAK13CS. The PCR of pC13J from yesterday is good! <br />
<br />
|tech= We ran colony PCR of pAK13CK on gel and nothing appeared. This could be because we used a janky Vent protocol. We miniprepped liquid cultures of pC13CK. A digest screen was run on the minipreps, but they did not look good. We will redo cloning for the construct. In other news, we got pC13BN transformants! We ran a colony PCR and grew up liquid cultures of pC13BN. The colony PCR looks good, and we will miniprep liquid cultures of pC13BN tomorrow. We made electrocompetent DH5a stocks, which are marked with blue circles with a line through it. Lastly, we double digested pC13Z and pC13AK with Xba, PstI, Sac, and ApaLI since we were running low on insert DNA. The gel for we ran for the double digestion looked good since we got 4 distinct bands for ''GFP'' and ''mRFP'' each. Next, we column purified pC13Z and pC13AK double digests and stored them in the freezer for later use. Today we attempted the first day of protoplasting and transforming <i>G. lucidum</i> in the Turgeon Lab. We used both our 3 day old and 1 day old cultures of <i>G. lucidum</i> in this attempt. Our initial pellet of both cultures appeared about the same size, though the younger one may have been slightly larger. We observed that the cells didn’t look like protoplasts, as they weren’t round and were smaller/very few in number. We looked at the solution after filtering and were able to see protoplasts, so it appears that something didn’t work with buffer/centrifuging. We miniprepped and made glycerol stock of the overnight cultures from yesterday--pC13I, pC13AF and pC13CV. We then digested these products. We electroporated for pAK13CS and plated the culture on Ampicillin plates. In addition, the PCR of pC13J worked! The transformations for pC13BC and pC13BV were too efficient, so we had to restreak them out to single colonies.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 1st<br />
|text= We are putting ''PgpdA'' with geneticin resistance, PtrpC with geneticin resistance, and PtrpC with hygromycin resistance between the ''lox'' sites. We miniprepped pC13BN. A Vent colony PCR of pC13CB colonies 1-5 was run to confirm successful cloning. We finished our attempt at protoplasting, once again unsuccessful, likely due to contamination on the fungal cultures. We ran a digest screen for pC13I, pC13AF and pC13CV, but only pC13AF (colony #2) appeared to work. We then ran a PCR with Vent for pC13I, pC13AF, pC13CV and pC13L, but they all failed. We transformed the new ligation for ''pC13CE'' into <i>E. coli</i>.<br />
|tech= We ran a PCR for amplification of pC13Y, pC13BQ, pC13BR, and pC13CD to put between the ''lox'' sites. The PCR products were then cleaned and quantified. We redid the ligation of pC13D with pC13CJ to form pC13CK. We electroporated the ligation into DH5a. We miniprepped the pC13BN 2 cultures. We then ran a Vent colony PCR of pC13CB colonies 1-5. We also grew up 5ml cultures of pC13CB colonies 1-5 and left them in the incubator overnight. We finished our attempt at protoplasting <i>G. lucidum</i> with the help of members of the Turgeon lab, with hygromycin as our selection marker, and we left the plates in the Turgeon lab incubator. We then combined our three blended cultures and blended again to make a new culture for tomorrow. We planned out a protoplasting schedule for this week to try running the protocol under different conditions since it takes a long time to get results. We believe there might be bacteria growing in the fungal cultures we are using for protoplasting since phosphinothricin does nothing to bacteria and we could not blend the fungi under sterile conditions. We will put ampicillin in later cultures to kill bacteria, which shouldn’t affect <i>G. lucidum</i> because only bacteria will have peptidoglycan cell walls. We did a PCR clean up for pC13A. We ran a digest screen for pC13I, pC13AF and pC13CV, but only pC13AF (colony #2) appeared to work. We then ran a PCR with Vent for pC13I, pC13AF, pC13CV and pC13L, but they all failed… <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 1st<br />
|text=Lydia's back too! Discussed what we have been doing for the past few weeks and immediate goals. <br />
|author=Rebecca<br />
}}<br />
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<div id="week12entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 12</h3><br />
<h5 class="centered">(09/02 - 09/08)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 2nd<br />
|text=Miniprepped ''PtrpC'' + ''T7 polymerase'', the ''PpelA'', the ''Anderson promoter'', and the ''Anderson promoter'' + ''T7 polymerase''. We are preparing to send pC13BN to sequencing. A Vent PCR was done on pC13CB to verify successful cloning. We began a new transformation attempt, now with fresh solutions to work with. However, this also seemed unsuccessful. We also ran a Vent PCR with the addition of MgSO4 for pC13AF, pC13CV, pC13I and pC13L minipreps--they all failed. Another Vent PCR was run to check inserts in pC13BC, pC13BV, and pC13CE, but resulted in no bands.<br />
|tech= We got colonies for pAK13CK and ran a colony PCR. The PCR didn’t show anything, but the primers are not ideal, so we will miniprep and digest screen. We digested PCRs of pC13BR, pC13BQ, pC13CD, and pC13Y with KpnI, HindIII. They were all cleaned. We digested miniprep of pC13BN with KpnI and HindIII. This was cleaned and dephosphorylated. We prepped pC13BN miniprep for sequencing with primer 31. Miniprepped pC13AY, pC13BF, pA13AM, and pA13AW cultures and will quantify tomorrow. We prepared pA13DC colonies 3 and 4 and pA13DB colonies 1 and 4 for sequencing Tuesday morning. We then reran a Vent PCR for pC13CB and grew up liquid cultures of the same colonies. We ran a gel for pC13A which showed that the PCR failed. We then redid a Vent PCR with the addition of MgSO4 for pC13AF, pC13CV, pC13I and minipreps of pC13L. We made 5 pAh13G cultures from glycerol stocks, and we electroporated pC13BJ, pC13BK, pC13BL, and pC13BM to make glycerol stocks. All transformations except pC13BM arced, but we are going to plate them anyway. We also attempted protoplasting again, using fresh enzyme osmoticum and STC. We couldn't really see mycelium at the beginning, and we could not see cells when we attempted to count them, possibly due to our mycelium being too old or having depleted its media. We made a new culture with a blended cell mass and ampicillin. We also made more CYM plates and media, and prepared a plan to test the sensitivity of <i>G. lucidum</i> to antibiotics and antifungals.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 3rd<br />
|text= Digest screen of ''PtrpC'' + ''T7 polymerase'' and the ''Anderson promoter'' + ''T7 polymerase'' showed good expected banding pattern. The pC13CK construct is correct. A gel of pC13CB PCR products was run and was inconclusive. We repeated the PCR from yesterday for pC13AF, pC13CV, pC13I and pC13L but using the Q5 polymerase. Except for pC13AF from colony #2, all else failed. We attempted to protoplast again, this time with emphasis on determining what our issue is in hopes that we can fix it.<br />
|tech= We quantified digestions and ligated pC13BQ + pC13BN, pC13Y + pC13BN, pC13BR + pC13BN, and pC13CD + pC13BN. We electroporated the ligations. We miniprepped pC13CK cultures and ran digest screen, which was successful. The construct is correct. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin'', which was unsuccessful. The pC13AY, pA13AM, and pA13AW preps were digested with EcoRI and PstI and run on a gel. Results indicate that pC13AY and some of the pA13AW colonies worked. The gel of Vent PCR of pC13CB did not turn out too well. We got a bunch of lanes with smeared rather than distinct bands. We then ligated and transformed ''T7 promoter'' with ''mRFP'' to obtain pAK13AO and ''PgpdA'' with ''GFP'' to create pC13CF. We ran a Q5 PCR and gel for pC13AF, pC13CV, pC13I and pC13L. Sadly, they all failed except pC13AF from colony #2. We are concerned with what’s going on. The PCR for pC13A failed again too. We miniprepped our pAh13G cultures. Our new fungal culture doesn’t have nearly as much mass in it, leading us to believe that the old cultures were almost definitely contaminated with bacteria. Our transformed pC13BK and pC13BM grew into masses covering the plates rather than colonies, and the others didn’t grow. We restreaked pC13BK and pC13BM hoping for single colonies. We attempted a diagnostic protoplasting, determining that pellets formed are likely from contaminations, as the new cultures with ampicillin did not produce pellets. We also tried to manually extract mycelium and enzyme digest it, but microscope visualization did not show signs of protoplasting having occurred. Even after filtering through one layer of cheesecloth and nylon, there was no appearance of protoplasts.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 3rd<br />
|text=Christine, Rebecca and Sara met up with Dr. Land again and we learnt more about setting up PID controls. Went to the lab next where Christine and Sara set up the heating element circuit, Rebecca soldered pins onto the arduino and Lydia drew up the circuit design.<br />
|author=Rebecca<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 4th<br />
|text= Transformed the ''PpelA'' and ''Anderson promoter'' + ''T7 polymerase'' into pSB1C3 as well as digest and ligate other promoters. The pC13BN construct was incorrect, so we will have to redo all of the Gibson transformations for the ''lox'' constructs. On a brighter note, sequence confirmation of pA13DB and pA13DC came back today!<br />
<br />
|tech= We got transformants for all ''lox'' constructs. We got sequencing back for pC13BN and it was incorrect, so we will redo the Gibson transformation and all the subsequent ''lox'' constructs. We also electroporated pC13BF and pC13AZ and digested pC13F and pA13AW with XbaI and PstI for ligation to make pC13AZ. pC13F and pC13BE KpnI and BamHI digests were then ligated to make pC13BH. pA13DB colony 1 and pA13DC colony 4 were sequence confirmed today. Lastly, we made cultures from restreaked pC13BM and pC13BK plates.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 5th<br />
|text= Today in lab we gel screened a bunch of old minipreps to see if they were still viable and made cultures of the ''Anderson promoter'' + ''T7 polymerase'' in pSB1C3. We digested the ''lox'' site constructs in order to ligate with pC13BN. In a meeting with Professor Turgeon we decided that we would leave <i>G. lucidum</i> behind for now and work with <i>Cochliobolus</i>, which her lab transforms regularly and has more expertise with.<br />
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|tech= The Gibson was digested with DpnI and electroporated into DH5a. We are growing DH5a for electrocompetent stocks. We did a PCR of pSB1C3 to append cut sites in order to ligate with ''holin''. We digested pC13Y, pC13BQ, pC13BR, and pC13CN for ligation with pC13BN and made cultures of three pC13AZ colonies. Gel screen of pC13F and pA13AW appeared successful and were then column purified. We also gel screened pC13P miniprep, SpeI and PstI digested pC13P, PCR of ''PgpdA'' for homologous regions, PCR of pC13F for homologous regions, and BamHI/KpnI digested pA13BE to verify the viability of old minipreps. We then amplified ''T7 polymerase'' from pC13E. We did a Q5 PCR for pC13CV, pC13I and pC13L. Our plates from transformation on Saturday showed conidia growth on the controls with no hygromycin. None of the plates with hygromycin showed any growth. We decided that we will now use <i>Cochliobolus</i> to protoplast and transform after meeting with Professor Turgeon again. To determine if the DNA is in the fungal genome and stable, we will plate on non-antibiotic and antibiotic plates, then replate transformants from the antibiotic plate onto non-antibiotic plates and back onto antibiotic plates. Since protoplasting is failing, we will turn to agrobacterium for <i>G. lucidum</i> transformation in the future. We will also check for homology between the promoter gpd in <i>G. lucidum</i> and <i>Cochliobolus</i> to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after <i>Cochliobolus</i> gpd by cutting into that region in the genomic promoter to transform. We also created glycerol stocks of pC13BM and pC13BK cultures and miniprepped them to submit for sequencing. We set up new cultures of colonies from transformation of pC13BC, pC13BV, and pC13CE, and ran colony PCRs of them (using our brand new tube of Q5 polymerase!) to check for inserts.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 6th<br />
|text= Most of old minipreps were no good and the ''PpelA'' transformation failed. The ''Anderson promoter+T7 polymerase'' gel results looked good and were submitted for sequencing. Oh no, the PCR of pC13CV, pC13I and pC13L failed again--we suspect something is wrong with our reagents. Use the new dNTPs from now on!<br />
<br />
|tech= We digested, ligated, and transformed to create pC13DD. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin''. Gel results indicate that pC13P miniprep, pC13P SpeI/PstI digests, ''PgpdA'' PCR for Gibson and the pA13BE BamHI/KpnI/SacI digest are not viable, but the pC13F PCR for Gibson might be okay. pC13BF transformation failed as well. The pC13AZ cultures were digest screened; results were good, so they were sent for sequencing. pC13P was plated and pC13E was cultured. We submitted pC13AF (colony #2) for sequencing. The Q5 PCR from yesterday--pC13CV, pC13I and pC13L--was a failure. We suspected that something is wrong with our reagents. Use the new dNTPs from now on! We ran PCRs to prepare the necessary parts for our Gibson assembly to produce parts with flanking <i>G. lucidum</i> homologous regions. We spun down the cultures of pC13BC, pC13BV, and pC13CE and stored them in the fridge.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type= anim<br />
|head=September 6th <br />
|text= We met at Flora Rose house to decide on an outline of the presentation and also decide a timeline.We met for the first time to discuss a preliminary outline for the presentation. Hannah will be handling all the human practices elements, Eric will be explaining relevant biological background and strategies, and Arun will be presenting all of our characterization data (hopefully!). We made a detailed structure which will include an introduction about styrofoam pollution and ecovative. Then we will move on to a brief overview of our wetlab work. It was clear that the wetlab portion should be divided into 3 main components. The components would be fungal toolkit, carotenoid pathway, and antifungals. Finally, we will end with results, human practices, and acknowledgments to all our beautiful advisors and sponsors.<br />
|tech= <br />
|author=Arun<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 7th<br />
|text= Digested and ligated the ''Anderson promoter+T7 polymerase'' into pSB1C3 and pSB2K3 as well as start cultures from glycerol stocks and transformation and gel screened the ''Anderson promoter'' + ''T7 polymerase''. We transformed ''afp1'' and ''Cht1_2''. For fluorescence, we electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ to obtain more plasmid DNA. We redid a Q5 PCR for pC13CV, pC13I, pC13AF and pC13L with the new dNTPs--PCR products were all bad except for pC13L.<br />
<br />
|tech= We did Q5 PCR to amplify pSB1C3 for Gibson with ''afp1'' and ''Cht1_2''. 3uL of PCR product was run on the gel, found to correct, and was cleaned. We ran a Gibson of pSB1C3 with both pC13CL and pC13BA. Both of these were transformed, ''Cht1_2'' into DH5a (time constant 4.82) and ''afp1'' into BL21 (time constant 4.84). We made glycerol stock of pC13BN. We ran a gel of pSB1C3 PCR from yesterday, which was successful, and performed a PCR clean up. We digested pSB1C3 and pC13CN with HindIII and BglII. We ligated these together to create pC13CO and transformed it into DH5a (time constant 4.82). We miniprepped pC13BN culture and did a double digest on pC13BN culture with KpnI and HindIII to see if internal cut sites were present. There was some digestion that occurred when it was run on a gel, so we are confident the sequence is correct. We double digested pC13BN again with KpnI and HindIII. We ligated it with pC13Y (time constant 4.86), pC13BR (4.86), pC13BQ (4.90), pC13CD (4.80). We got transformants for pC13DD. A colony PCR was run and pC13DD colony 3 looked good. This colony was grown in liquid culture for miniprepping and sequencing. We grew cultures of pC13BX, pC13CK, and pC13BN for submission (pC13BX submitted directly; pC13CK and pC13BN need sequence confirmation first). pC13BF transformants were cultured, pC13AZ cultures were digested with EcoRI/PstI and gel screened; results were good, so they were submitted for sequencing. Digested pK13AL and pA13AW with XbaI/PstI for ligation to make pK13AX. pC13P and pC13E were digested with EcoRI/PstI and ligated to make pC13AY. Cultures of pC13Q and pC13BE were made from glycerol stocks. We electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ. We also ran a Q5 PCR of pC13CB and pAK13AO using standard BioBrick primers 23 and 24 in order to confirm lengths of inserts. We redid the Q5 PCR with the new dNTPs for pC13CV, pC13I, pC13AF and pC13L--pC13CV, pC13I and pC13AF were bad, but pC13L was good! We suspect that electroporation did not go well to begin with. We reran the 2-step Q5 PCR for pC13A. We ran a gel of our PCR to create Gibson assembly parts, and it appeared unsuccessful. We ran the colony PCRs of pC13BC, pC13BV, and pC13CE on a gel, and only observed bands for a few pC13BC colonies.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=September 7th<br />
|text= Five team members went to the Ithaca Sciencenter and taught a group of 18 children and 17 parents about the power of DNA.<br />
|author=Hannah<br />
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{{:Team:Cornell/templates/nbentry<br />
|type= anim<br />
|head=September 7th <br />
|text= After researching we found out that the powerpoint presentation should be centered around design and images rather than content. We decided to meet tomorrow to discuss possible design layouts and schemes. We looked at many examples of presentations and considering our project, we think we should have a very modern and green theme. Arun will explore with this later on.<br />
|tech= <br />
|author=Arun<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 8th<br />
|text=We met super early in the morning (10 am!) for brunch and some delicious cake. We also got work done and made an entire storyboard for the presentation. Our golden (or perhaps orange) idea of the day was a growing carrot representation for the carotenoid pathway. <br />
|tech=<br />
|author=Eric, Arun <br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 8th<br />
|text= We miniprepped ''PpelA'', PtrpC, and ''TtrpC''. We got transformants with ''afp1'' and ''Cht1_2'' and for all of the ''lox'' site constructs. However, only the pC13CO looked good based on a gel screen. We will sequence the ''lox'' construct. We also miniprepped pC13CV, pC13I, pC13AH and pC13AG. <br />
<br />
|tech= We got transformants from the ''afp1'' and ''Cht1_2'' Gibsons. There were a lot of red colonies and very few white ones, and an attempted pick and streak of the white colonies was performed. We got transformants for pC13CO, pC13BN + pC13CD, pC13BN + pC13Y, pC13BN + pC13BQ, and pC13BN + pC13BR. A colony PCR was performed, and a gel of the PCR products looked good for only pC13CO colony 2. No positive inserts were present, so maybe the ''lox'' construct is not correct. To check, we will sequence it tomorrow. Afterwards, we miniprepped pC13BF, pC13P, and pC13Q and digested pC13BF. We ran the gel for pC13A and did a PCR clean up for it, as well as for pC13J and pC13L. We also digested pSB1C3 with XbaI and PstI, and miniprepped pC13CV, pC13I, pC13AH and pC13AG. We attempted to run another gel of our PCR product to create Gibson assembly parts, which looked even worse than yesterday. We also did a PCR cleanup of pC13BJ, pC13BK, pC13BL, and pC13BM.<br />
|author=Rafael<br />
}}<br />
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<h3 class="centered">Week 13</h3><br />
<h5 class="centered">(09/09 - 09/15)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 9th<br />
|text= PpelA preps were quantified and the <i>G. lucidum</i> promoter was amplified and gel screened. We are preparing our pC13CO, pC13BA, and pC13CL cultures to sequence. The correct insert is in pC13BA based on a gel screen. We digested pC13L and dephosphorylated the pSB1C3 digest from yesterday. We prepared an overnight culture of <i>Cochliobolus</i> and necessary solutions for a protoplasting attempt tomorrow. <br />
|tech= We ran a digest screen on pC13F with KpnI (mRFP has 2 internal KpnI cut sites). No digestion occurred since the enzyme was degraded. We submitted sequencing for pC13CK and pC13BN. We miniprepped pC13CO, pC13BA, pC13CL cultures and need to quantify and prep for sequencing. We ran a colony PCR on streaked pC13BA and pC13C plates and got a definite correct insert on pC13BA, and a lot of nonspecific amplification on pC13CL. We will run a digest screen and sequence. pC13BF colonies were quantified and gel screened and ''PgpdA'' was amplified from <i>G. lucidum</i> DNA, digested, and gel screened. The results were good, thus it will be Gibson assembled. We redid the PCR for pC13A with new materials but it unfortunately failed again. We also digested pC13L with XbaI and PstI and dephosphorylated the pSB1C3 digest from yesterday. We made an overnight culture of <i>Cochliobolus</i> in the Turgeon lab, as well as preparing fresh solutions of enzyme osmoticum and regeneration medium. We miniprepped the cultures of pC13BC, pC13BV, and pC13CE from the weekend. We added antibiotics to CYM plates, and intended to inoculate them for the growth experiment for testing <i>G. lucidum</i>’s antibiotic sensitivity, but the plates were too wet for reliable inoculation (that would spread in a circle of constant diameter), and inoculation was postponed so the plates could dry.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 9th<br />
|text=We met with our design master Oat to discuss design considerations for the presentation. Oat will be making many designs, including a kill switch diagram, homologous recombination figure, our carotenoid image, a toolkit representation, and of course lots of mushrooms. <br />
|tech= <br />
|author=Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 10th<br />
|text= ''Anderson promoter'' + ''T7 polymerase'', in pSB1C3 and BBa_J61002 were miniprepped, glycerol stocked, and prepared for biobrick submission. For fluorescent constructs, a Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. The pC13BN construct was incorrect. An overnight culture was prepared for ''rbs'' + ''crtI'' (from pC13L) at 4C. We made our first attempt at protoplasting and transforming <i>Cochliobolus</i> with the help of the Turgeon Lab. We set up cultures of pC13X and pC13AV in preparation for part submission. We started the growth experiment for testing <i>G. lucidum</i>’s antibiotic sensitivity.<br />
<br />
|tech= We prepared pC13CO, pC13CL, pC13BA for sequencing. We made glycerol stocks of pC13CL and pC13BA. We got a sequence confirmation for pC13CK. We got sequencing for pC13BN and had a portion of the lac operon inside the prefix and suffix...a weird recombination must have occurred. pC13AZ sequencing results were good! That and pA13AM and pA13AW were miniprepped for glycerol stocks and biobrick submission. A PCR to add homologous regions to pC13F for Gibson assembly was done. The gel of Q5 PCR of pAK13AO colonies 1,2,3 and pC13CB 1,3,4 all had correct lengths. Thus, we miniprepped the colonies with correct plasmid insert lengths and submitted them for sequencing. We ran a PCR and electroporation for pC13CV and pC13I, which turned out to be bad. We made overnight E. Coli culture for pC13AF (colony #2). An overnight ligation was done for ''rbs'' + ''crtI'' (from pC13L) at 4C. We ran a PCR to check the inserts in pC13A, pC13I and pC13J. We protoplasted and transformed <i>Cochliobolus</i> with Dongliang, a post-doctoral research associate in the Turgeon Lab. We used our constructs of pAh13G / pC13BR linearized with SphI, as well as the highly efficient <i>Cochliobolus</i> vector PNG from the Turgeon lab as a "positive" control. We submitted pC13BM 1 and pC13BK 1 for sequencing. We homogenized a dilute culture of <i>G. lucidum</i> and inoculated each of the plates for the antibiotic sensitivity test with 20 μL aliquots, without spreading.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 10th<br />
|text=Oat, Eric, Arun, and Hannah sat down to make figures for the presentation. Arun also chose his first color scheme: a baby blue flanked by a serene green. We got many designs done including the carotenoids, homologous regions, kill-switch, and the construct pieces.<br />
|tech=<br />
|author= Arun, Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 11th<br />
|text= Redid transformations of ''PpelA'' and the ''Anderson promoter+T7 polymerase''. We sent the ''Anderson promoter'', pC13CO, pC13CL, and pC13BA to sequencing. Gel electroporation for pC13I showed that it was good, but nothing for pC13J. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists in iGEM! We continued with protoplasting <i>Cochliobolus</i> in the Turgeon Lab, and we ran a Gibson assembly of homology regions in <i>G. lucidum</i>.<br />
<br />
|tech= We submitted pC13CO, pC13CL, and pC13BA for sequencing. After, we miniprepped and made stocks of pC13AY and pC13AZ. Also, we submitted pA13AW for sequencing. Transformation of pK13AX was unsuccessful because the wrong resistance plate was used. pC13BF and pK13AX were transformed today. We miniprepped the overnight culture of pC13AF. Gel electroporation showed that pC13I samples are good, but none of the pC13J samples are positive. We ran a PCR for the inserts in pAK13CR. We were surprised to know that ''rbs'' + ''crtE'' (pC13J) already exists! We made 1000x hygromycin and 100x geniticin. We checked plates from our protoplasting and transforming attempt under the microscope. It appeared unsuccessful so next time we will use all the protoplasts, at least 300 µL. We plated a layer of cym agar and the respective antibiotic on top of the regeneration agar gel as well as inoculating 4 new plates of <i>Cochliobolus</i>. We also ran our Gibson assembly of homology regions in <i>G. lucidum</i>. We miniprepped pC13X and pC13AV in anticipation of part submission, and ran a PCR (to check for inserts) from minipreps of pC13BC, pC13BV, and pC13CE, as well as pC13X and pC13AV (just in case).<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 12th<br />
|text= Ran Gibson assembly for the <i>G. lucidum</i> promoter into pSB1C3. pC13BA is now a BioBrick! We also began cloning a construct with both fluorescence and resistance genes by ligating pC13BR and pC13AT. We successfully electroporated ''E.Coli'' with pC13J! We digested pAK13CR, and then we ligated ''rbs'' + ''crtB'' into pSB1C3, ''crtE'' into pSB1C3. After finding our Gibson assembly from last night left in the water bath, we attempted it again as well as preparing some of our Biobricks for submission. We checked pC13BC, pC13BV, and pC13CE and prepared the successful constructs for sequencing.<br />
<br />
|tech= We prepped pC13BX and pC13BA for BioBrick submission and got sequencing back. pC13BA was correct, and pC13CO and pC13CL were incorrect. We redid the Gibson for pC13CL and transformed it. We redid pC13CO digestions, ligation, and transformations. We transformed the redone ''lox'' Gibson from yesterday. We streaked out CYM plates with <i>Aspergillus Niger</i>, ran the Gibson assembly for ''PgpdA'' in pC13F, and transformed. We also digested pC13BR with SpeI and PstI and digested pC13AT with Xba and PstI, ran a gel of digestions to verify lengths, and column purified the inserts from digestion. We did an electroporation with pC13J and it was successful! We ran a gel for pAK13CR for confirmation and then we did a PCR clean up. We digested pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, and then we ligated ''rbs'' + ''crtB'' (from pAK13CR) into pSB1C3, ''crtE'' (from pC13I) into pSB1C3. We prepared pC13BJ pC13BK pC13BL pC13BM and pC13BR for Biobrick submission. It seems our Gibson assemblies from yesterday were left in the water bath all night, so we reran them (pC13BK + pC13BM + pC13CD, pC13BQ, or pC13Y) before we transformed and plated the new assembled plasmids. We made a culture of pC13CG from glycerol stock. We ran a gel of the PCRs of pC13BC, pC13BV, pC13CE, pC13X, and pC13AV - there were correct bands for at least two of everything, and successful pC13BC, pC13BV, and pC13CE constructs were prepared for sequencing. We also set up a culture of pC13BQ in anticipation of part submission.<br />
<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 13th<br />
|text= Gibson assembly for the <i>G. lucidum</i> promoter yielded no colonies. Numerous ligations and electroporations were done for fluorescent constructs. We are starting the ''afp1'' <i>Aspergillus</i> growth assay. We did electroporation for pC13I, pC13CV and pAK13CS but pAK13CS arced. We prepared numerous Biobricks for submission to iGEM HQ.<br />
<br />
|tech= We submitted pC13BC, pC13BV, and pC13CE for sequencing. We reinoculated cultures of pC13BN, pC13CL, pC13CO. Then, we conducted a preliminary test on activity of ''afp1''. We cut circles of various sizes from dialyzing membranes and dipped in grown ''afp1'' in BL21 culture. The membrane pieces were then placed on the fully grown <i>Aspergillus</i> plate. This preliminary test will see whether or not there is activity of ''afp1'' on the <i>Aspergillus</i>. We are hoping for a ring of death on the <i>Aspergillus</i> plate. Unfortunately, there were no colonies for the ''PgpdA'' assembly. We then ran a gel for some of the remaining assembled DNA and no bands appeared. We also digested pC13AU with Xba and PstI to ligate downstream of pC13BR and pC13BQ. We then column purified pC13BQ, pC13BR, pC13AT, pAK13AO, pC13AU, pC13F. After, we dephosphorylated pC13BQ, pC13BR, pC13F, and ligated pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, and pAK13AO with pC13F. Next, we electroporated all ligations into both BL21 and DH5α cells. Hopefully, a fluorescence trial can be done tomorrow with these colonies! We made overnight culture for pC13J. We did electroporation for pC13I, pC13CV and pAK13CS. The last one unfortunately arced. Our Gibson-transformed plates had too many colonies so we restreaked them. We miniprepped pC13CG and prepared it for Biobrick submission. We also checked our plates in the Turgeon lab. The control plate is growing well, and the others appear to have something very small starting to grow. We will give them more time before checking again. We also received our sequencing results from pC13BM 1, pC13BK 1, pC13BC, pC13BV, and pC13CE, which all appear to have been successful. We set up a culture of pC13CD from its glycerol stock to re-miniprep and submit as a biobrick.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 13th<br />
|text= Managed to get the arduino to control the heating circuit! <br />
|author=Rebecca<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 14th<br />
|text= The ''Anderson promoter'' + ''T7 polymerase'' were electroporated, but both ligations arced, so transformations are very likely to not succeed. We are unsuccessful with pC13CL and pC13BN. We miniprepped pC13J cultures. <br />
<br />
|tech= Electroporated pK13AX, but both arced, so outlook is grim. We also picked 12 colonies from potential pAK13CY colonies and grew corresponding colonies in liquid culture. We then prepped pC13AQ, pC13AT, and pC13AU for biobrick submission. Later that day, we picked colonies for pC13BN and pC13CL from plates from Thursday, did a colony PCR, and grew cultures for each. We re-electroporated the pC13CO construct, and plated four different versions of it. We ran a gel of all 14 colonies of pC13BN and pC13CL. The pC13CL one came out blank and the pC13BN had bands, but not where they were expected. We miniprepped pC13J and made glycerol stock. We ran PCR to check the following inserts: pC13AF, pC13P, pC13CV, pC13I and pC13BG. We ran colony PCRs on the restreaked plates containing our Gibson assembly products of homology regions. We miniprepped pC13BQ and pC13CD for part submission.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 15th<br />
|text=We once again got brunch and ate some school famous chocolate peanut butter cake. We agreed that Becker brunch has the best brunch. Then we sat down all day and put together the majority of the presentation including graphics and content. In addition, Hannah and Eric started writing the script for their part of the presentation. Arun will start writing his script as soon as characterization data starts coming in.<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 15th<br />
|text= We are continuing cloning of ''Lambda Holin'', ''chitinase'', and the ''lox'' sites and adding homologous regions to the ''lox'' sites for Gibson. We started the ''afp1'' assay with <i>Aspergillus</i>. We did a digestion for pC13AF and pC13P, and redid the ligation for insert in pC13L and vector pSB1AK8.<br />
<br />
|tech= We plated 100μL of ''afp1'' culture on fresh <i>Aspergillus</i> plates to demonstrate its effectiveness. We ran a PCR for pC13AI with primers 45/52 and 48/51 to add homologous regions for Gibson. The PCR protocol was a 1-step PCR with a 60C annealing temperature and 1:10 extension using GC buffer. We picked colonies from new pC13CO plates. We miniprepped pC13CL colonies 3 and 4, and pC13BN colonies 5 and 6 although the results of the gel was not clear. We redid the PCR for pC13BN. We also redid the PCR to append cut sites to pC13F for creation of pC13BF. We did a PCR clean up for the inserts in pC13BG, pC13P and pC13AF. We ran a PCR for pC13J and pC13BG. We prepared a double digestion for pC13AF and pC13P, and redid the ligation of ''rbs'' + ''crtI'' (from pC13L) and vector pSB1AK8. We ran gel of the colony PCR of our Gibson products, looking 4-5kb fragments. It appears that pC13BK + pC13BM + pC13BQ colony 1 was the only possibly successful reaction, so we made cultures of it, as well as various other cultures. We prepared pC13BC, pC13BQ, pC13BV, pC13CD, and pC13CE for part submission.<br />
|author=Rafael<br />
}}<br />
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<div id="week14entry" class="nb-week"><br />
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<h3 class="centered">Week 14</h3><br />
<h5 class="centered">(09/16 - 09/22)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 16th<br />
|text=Compared with last week’s jigsaw induced excitement, this week was relatively tame. With all of the necessary pieces in our possession, we set out to assemble our incubator. After sifting through the leftover drylab supplies from last year’s project, we came up with a couple tubes of silicone adhesive that we could use. Insulation pieces were adhered to their respective plastic backings and were allowed to dry for a couple days. <br />
|author=Mac<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 16th<br />
|text=We started taking measurements of <i>G. lucidum</i> growth on CYM plates by tracing the central mass of fungus--the differences between the plates are really interesting! Sequencing for pC13AY was successful so we prepared more culture. We did gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG, but only pC13J colonies are positive. We then did digestions for the following vectors: pC13BG, pC13AG, and pC13KI. We still do not have any growth on antifungal plates.<br />
|tech= We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of <i>G. lucidum</i> growth on CYM plates (plated on September 10) through tracing the central mass of fungus--the differences between the plates are really interesting, but there’s a small contamination on one plate. We transplanted mycelia from the G418 plates. We are slightly concerned that there’s still no growth on the antifungal plates. We submitted pC13CL and pC13BN for sequencing. We ran the gel for the colony PCR of pC13CO but they all failed. We will repick the white colonies from pC13CO plates. We had successful sequencing for pC13AY and we made cultures for glycerol stocks. We also retired the PCR to append cut sites to pC13F using a higher concentration of dNTPs and began a digestion of pA13BE in order to gel extract ''PpelA''. We electroporated ''E. Coli'' with pAK13CS, which unfortunately arced again… We performed gel electrophoresis for inserts in pC13CV, pC13I, pC13J and pC13BG. Only pC13J colonies are positive. We also did digestions for the following vectors: pC13BG with SpeI and PstI, pC13AG with EcoRI-HF and XbaI, and pC13K with EcoRI-HF and XbaI.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 17th<br />
|text=We tried transformation of <i>Cochliobolus</i> again. We ran a PCR to append cut sites to pC13F but no band was shown on a gel. We redid the Gibson of ''Cht1_2'' and pSB1C3. When we ran a PCR fo pC13AI, weird band patterning was observed. We purified the vectors we digested yesterday: pC13BG, pC13AG and pC13K. In addition, we purified inserts of pC13AF and pC13P. We continued recording measurements of <i>G. lucidum</i>’s growth in the presence of antibiotics.<br />
<br />
|tech=We attempted transforming <i>Cochliobolus</i> with pC13BR, pC13CD, pUCATPH and pC13AY. We ran a gel of the pA13BE digest and extracted ''PpelA''. We also reran a PCR to append cut sites to pC13F again using a different tube of dNTPs and re-diluted primers. We ran a colony PCR and screened it on a gel, but no bands appeared. Some of the cultures were miniprepped anyways, and they will be screened for presence of the insert (~340bp). We detected some growth on the afp1 assay plates. We redid the Gibson of ''Cht1_2'' and pSB1C3 PCR. We electroporated this Gibson into DH5a cells and plated it on chloramphenicol LB plates. We reran the PCR of pC13AI with primers 45/52 and 48/51, annealing temperature of 61.5C and extended for 30s. We did a double digest of pC13CO with EcoRI-HF & PstI. We ran a 1% TAE gel of pC13AI PCR products for 30 minutes at 100V. The bands for pC13AI with 45/53 were seen at 1.5kb and 1.1kb; bands for pC13AI with 48/51 were seen at 1.5kb and 1kb. We still have a weird banding pattern. We may need to do a gradient PCR to find a better temperature for the primers and to test the PCR without the GC buffer. We purified and dephosphorylated the vectors we digested yesterday--pC13BG, pC13AG and pC13K. We also purified inserts of pC13AF with EcoRI-HF and SpeI, and pC13P with EcoRI-HF and SpeI. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 17th<br />
|text=We met for our first run through of our presentation. The script needs some work, but it’s coming along nicely. It seems that the only part lacking in the presentation is the characterization data and charts. They coming soon!<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 18th<br />
|text=We are getting more data with regards to fungal growth on resistance plates! We ran the pC13CO digests on a gel and they looked good. We ran a PCR of pC13AI to amplify ''lox'' sites for Gibson.<br />
|tech=We used less hygromycin for transformation because Cochliobolus concentrations are less than <i>G. lucidum</i> concentrations. We are recording more and more data with regards with fungal resistance! We found a new contaminant on another plate but we will keep an eye out for it and make sure our measurements will not include its small area of contact with <i>G. lucidum</i>. We ran the gel extracted ''PpelA'' and pC13F PCRs on a gel but there were no bands. However, they may still be successful as the concentration were fairly low and may not have appeared on the gel. We ran the pC13CO digests on the gel and looked good. We ran a PCR of pC13AI to amplify ''lox'' sites for Gibson with harsh conditions to remove the nonspecific amplification. <br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 19th<br />
|text= Something may be wrong with the pC13AI plasmid based on gel screens. We ligated the constructs from Tuesday--''crtB'' with ''crtI'' and ''crtI'' with PtrpC. We continued recording measurements of <i>G. lucidum</i>’s growth in the presence of antibiotics.<br />
|tech= We quantified the holin minipreps. Both were around 48ng/uL, which is too low to send to sequencing. We will reculture and miniprep when LB is ready. We ran a PCR of pC13AI with primers 45/52 and 48/51. We ran the PCR products on a 1% gel and found bands at 1.5kb and 1.2kb for ''lox1'' and 1.5kb and 1kb for ''lox2''. There may be something wrong with the pC13AI plasmid. We also ran a 1 step PCR of ''lox1'' and ''lox2'' with a 59C annealing temperature. We submitted pC13J for sequencing with Primer 31. We also ligated the constructs from Tue--in short, ''crtB'' + ''crtI'', ''crtI'' + PtrpC. We ran a PCR with Q5 polymerase for inserts in pC13M, pC13J and pAK13CR and ran a gel for them--pC13J was good, pC13M had a faint band but nothing for pAK13CR. We took photos of the plates for the experiment on <i>G. lucidum</i>’s growth in the presence of antibiotics, to capture the differences in appearance in addition to colony size.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 20th<br />
|text= We will start a new growth assay to test afp1. We ran a Q5 PCR for pAK13CR.<br />
|tech= We began another growth assay because the previous ones showed no results. The cells may bave been dead by the time the cells reached them. This method of spreading the ''afp1'' culture like an antibiotic and plating mycelium smoothie on the top should give more consistent results. We retried electroporation with pAK13CS and it sadly arced again… We also ran a Q5 PCR for the insert in pAK13CR. We prepared and autoclaved CYM + agar, for making plates.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 21st<br />
|text= We set up the PCR to append KpnI and BamHI cut sites to pC13F again with different annealing temperatures and ran a gel to test the PCRs. Ran Gibson for pC13AI. A large Q5 PCR was done on fluorescent constructs pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. We ran the gel for pAK13CR from yesterday. We ligated the following: ''RBS'' + ''crtE'' to make pAK13CQ, ''RBS'' + ''crtB'' to make pC13CV, ''ctrB'' + pC13BG to make pC13DP, ''RBS'' + ''crtI'' to make pAK13CS. We attempted to transform ''pC13BV'' into BL21 <i>E. coli</i>, but failed. We then poured CYM plates.<br />
|tech= We ran a Gibson of the DpnI digest of pC13AI PCRs. In terms of characterization, we ran a huge Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F using standard biobrick forward and reverse primers 23 and 24. We set up the PCR to append KpnI and BamHI cut sites to pC13F again with different annealing temperatures and ran a gel to test the PCRs. We ran a gel for the PCR products--insert of pAK13CR--from yesterday, and then we did a PCR clean up of the inserts of pAK13CR and pC13I. We then did electroporation for pC13DJ and pC13BD. The latter arced... We digested pSB1C3 with EcoRI-HF and PstI, pSB1C3 with XbaI and PstI, pAK13CR with EcoRI-HF and PstI, pC13I with XbaI and PstI, pSB1AK8 with SpeI and PstI, pC13J with XbaI and PstI, pC13M with XbaI and PstI. We then dephosphorylated digested pSB1C3 and pSB1AK8. Ligation was performed for the following: ''RBS'' + ''crtE'' (from pC13J) into pSB1AK8 to create pAK13CQ, ''RBS'' + ''crtB'' (from pAK13CR) into pSB1C3 to create pC13CV, ''ctrB'' (from pC13M) into pC13BG to create pC13DP, ''RBS'' + ''crtI'' (from pC13L) into pSB1AK8 to create pAK13CS. We transformed ''T7'' + ''hph'' (pC13BV) into BL21 cells and plated on chloramphenicol plates. Unfortunately they arced, so we will need to repeat that with clean electroporation cuvettes. We poured more CYM plates.<br />
|author=Rafael<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 21st<br />
|text=We ran through our slides for the full team. We received a lot of good advice, and we still have a lot of work to do, but most of it will have to wait until after tuesday so we don’t fail our orgo exam! The main suggestion would be to change the theme of the presentation as the baby blue and serene green are too subtle to captivate attention. In addition, there are little glitches to the images.<br />
|tech=<br />
|author=Arun , Eric<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 22nd<br />
|text= We plated pC13AI. We are continuing <i>Aspergillus</i> growth assay, and completed the <i>G. lucidum</i> growth assay.<br />
|tech= We did a PCR cleanup of the pC13AI Gibson pieces, electroporated (time constant was 2.88 ), and plated the cells. We continued taking masses for <i>Aspergillus</i> with ''afp1'' growth assay and began a growth assay with inoculating discs. We measured the colonies of <i>G. lucidum</i> (control, and in the presence of various antibiotics) one final time, as they reached the maximum possible sizes for the petri dishes they were growing in.<br />
|author=Rafael<br />
}}<br />
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<div class="twelve columns"><br />
<h3 class="centered">Week 15</h3><br />
<h5 class="centered">(09/23 - 09/29)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 23rd<br />
|text= This week, the incubator box was completed. The fabrication and electrical groups came together to begin combining and testing. Initial tests with the temperature sensor yielded very positive results. We were able to successfully set and reach a target temperature with our sensor/feedback system. Unfortunately, our humidity sensor malfunctioned while we were running tests, however, we were still able to gather some data. Initial humidity tests indicate that our misting apparatus is able to produce a 12% increase in humidity over a time span of 8 minutes. Using a hotter water supply, as well as allowing the misting apparatus to run for longer would certainly allow us to increase humidity even more. <br />
|author=Mac<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 23rd<br />
|text= Today we ran a gel to verify insert lengths from Q5 PCR products of fluorescent constructs. We also transformed our T7 + hygromycin resistance construct into <i>E. coli</i> for characterization, and set up a zone of inhibition experiment to test the efficacy of antibiotics and antifungals on <i>G. lucidum</i>.<br />
|tech= Two gels, each with 20 wells, were run simultaneously for fluorescence. The DNA samples loaded were from a Q5 PCR on pC13BQ with pC13AT, pC13BQ with pC13AU, pC13BR with pC13AU, pC13BR with pC13AT, and pAK13AO with pC13F. pC13BV was transformed into electrocompetent BL21 <i>E. coli</i>. A culture of <i>G. lucidum</i> was diluted and homogenized, and 150 μL aliquots were spread on plates and let dry. Paper discs were soaked in 100X solutions of geniticin, hygromycin, ampicillin, kanamycin, and chloramphenicol and placed in the center of each plate. We miniprepped ''holin'' 8 & 9. The results from the growth assay do not look good. However, we found that the T7 polymerase in BL21 is controlled with an IPTG inducible promoter, so more pC13BA was grown with IPTG and plates were all re-inoculated with the corresponding amounts. An appropriate volume of LB was also added to some plates to account for mass changes. The zone of inhibition tests were also restarted with recultured (with IPTG) pC13BA. The pC13BN transformations yielded no colonies.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 24th<br />
|text=The gel of pC13AI showed nothing - the EtBr (for gel staining) needs to be replaced. We also continued putting our T7+resistances constructs into <i>E. coli</i> for part characterization.<br />
<br />
|tech=We miniprepped the pC13AI cultures. We amplified with primers 45-52 and primers 48-51 and ran a gel (expected size at 1.5kb). Nothing appeared on the gel, not even the ladder. The EtBr should be replaced. There were too many transformants for pC13BV in the BL21 strain, so those cells were restreaked onto a new plate. Additionally, we transformed pC13BC into BL21 as well, and characterize that part as well.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 25th<br />
|text= Sequencing of pC13CO did not turn out to be positive. Still trying to get data from ''Aspergillus'' and growth assay, and characterizing the ''T7 promoter'' and antibiotic resistances constructs. <br />
|tech= The sequencing of pC13CO came back as ''PtrpC'' + ''GFP''. We are not sure why or how. We either miniprepped the wrong thing or put the wrong thing into sequencing. We continued to take measurements for growth assays. We put a lot of <i>Aspergillus</i> around the inoculating discs for gathering more data. We set up cultures of BL21, with no plasmid and with pC13BC and pC13BV, and then standardized the optical densities of the cells between the three cultures. We spread 50 μL aliquots onto LB, adding discs dipped in 10X, 1X, and 0X concentrations of geneticin and hygromycin.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 25th<br />
|text=We made a lot of changes to the presentation tonight, and it is an almost polished form now. Arun switched to his next color scheme: a deep Cornell red with baby blue banners. Eric has gotten his part of the script memorized. He will be speaking about the three components of our project. The fungal toolkit, and its implementation through the carotenoid pathway and antifungals expression. Hannah has also prepared her part on human practices. In addition she has prepared an analysis on the market trends towards bioplastics.<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=September 26th<br />
|text=The ''T7 promoter'' and hygromycin resistance construct appears functional in <i>E. coli</i>.<br />
|tech=There was a clear zone of inhibition around the discs dipped in 10X hygromycin, no inhibition around the discs dipped in sterile water, for the wild type BL21 <i>E. coli</i>. However, there was normal growth around the 10X hygromycin discs for the BL21 <i>E. coli</i> transformed with our pC13BV construct. For the geneticin tests, the antibiotic appeared relatively ineffective, possibly due to degradation, and that experiment will need to be repeated.<br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 26th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. <br />
|tech=<br />
|author=Eric<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=pres<br />
|head=September 27th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. Arun also experimented with backgrounds of clouds, dirt, and toolkit to make the introduction slides very sharp. It look like we now have a solid design for our presentation.<br />
|tech=<br />
|author=Arun,Eric<br />
}}<br />
<br />
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<!-- OCTOBER<br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/templates/headerTeam:Cornell/templates/header2013-09-28T03:47:37Z<p>R.Lizarralde: </p>
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-09-28T03:36:11Z<p>R.Lizarralde: </p>
<hr />
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</script><br />
<div class="row team-bios-container"><br />
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<div class="ten columns team-bios-container"><br />
<div class="row""><br />
<div class="twelve columns"><br />
<h2 class="centered">Animation</h2><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
Our project was less amenable to fancy 3D animations this year, so we took a different approach: trying to pack a lot of information in minimally designed, cartoon-style animation.<br />
<iframe width="755" height="520" frameborder="0" src="https://dl.dropboxusercontent.com/u/496608/animation.html" scrolling="no"></iframe><br />
<br/><br />
<br/><br />
However, we also worked on other visual displays, such as a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness. Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration.<br />
<iframe width="755" height="520" frameborder="0" src="https://dl.dropboxusercontent.com/u/496608/animation2.html" scrolling="no"></iframe><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-09-28T03:34:41Z<p>R.Lizarralde: </p>
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<div class="ten columns team-bios-container"><br />
<div class="row""><br />
<div class="twelve columns"><br />
<h2 class="centered">Animation</h2><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
Our project was less amenable to fancy 3D animations this year, so we took a different approach: trying to pack a lot of information in minimally designed, cartoon-style animation.<br />
<iframe width="755" height="520" frameborder="0" src="https://dl.dropboxusercontent.com/u/496608/animation.html" scrolling="no"></iframe><br />
<br/><br />
<br/><br />
However, we also worked on other visual displays, such as a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness. Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration.<br />
<iframe width="755" height="520" frameborder="0" src="https://dl.dropbox.com/u/3824307/iGEM/youtube.html"></iframe><br />
</div><br />
</div> <br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-09-28T03:33:30Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_wetlab').addClass('active');<br />
});<br />
</script><br />
<div class="row team-bios-container"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/wetnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row""><br />
<div class="twelve columns"><br />
<h2 class="centered">Animation</h2><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
Our project was less amenable to fancy 3D animations this year, so we took a different approach: trying to pack a lot of information in minimally designed, cartoon-style animation.<br />
<iframe width="755" height="520" frameborder="0" src="https://dl.dropboxusercontent.com/u/496608/animation.html"></iframe><br />
<br/><br />
<br/><br />
However, we also worked on other visual displays, such as a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness. Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration.<br />
<iframe width="755" height="520" frameborder="0" src="https://dl.dropbox.com/u/3824307/iGEM/youtube.html"></iframe><br />
</div><br />
</div> <br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-09-28T03:31:19Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_wetlab').addClass('active');<br />
});<br />
</script><br />
<div class="row team-bios-container"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/wetnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row""><br />
<div class="twelve columns"><br />
<h2 class="centered">Animation</h2><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
Our project was less amenable to fancy 3D animations this year, so we took a different approach: trying to pack a lot of information in minimally designed, cartoon-style animation.<br />
<iframe width="755" height="500" frameborder="0" src="https://dl.dropboxusercontent.com/u/496608/animation.html"></iframe><br />
<br/><br />
<br/><br />
However, we also worked on other visual displays, such as a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness. Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration.<br />
<iframe width="755" height="500" frameborder="0" src="https://dl.dropbox.com/u/3824307/iGEM/youtube.html"></iframe><br />
</div><br />
</div> <br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/wetlab/animationTeam:Cornell/project/wetlab/animation2013-09-28T03:29:48Z<p>R.Lizarralde: Created page with "{{:Team:Cornell/templates/header}} <html class="no-js" lang="en"> <script type="text/javascript"> $(window).load(function() { $('li.pg-project_wetlab').addClass('active'); ..."</p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_wetlab').addClass('active');<br />
});<br />
</script><br />
<div class="row team-bios-container"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/wetnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row""><br />
<div class="twelve columns"><br />
<h2 class="centered">Animation</h2><br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
Our project was less amenable to fancy 3D animations this year, so we took a different approach: trying to pack a lot of information in minimally designed, cartoon-style animation.<br />
<iframe width="755" height="500" frameborder="0" src="https://dl.dropbox.com/u/3824307/iGEM/youtube.html"></iframe><br />
<br/><br />
<br/><br />
However, we also worked on other visual displays, such as a program that simulates mycelial growth using an image as a mapping of food concentration-- the mycelium grows fastest where the image is brightest, and consumes the brightness. Although this didn't always make the prettiest images, we were able to adapt the program to produce excellent animations by breaking away from modeling the food concentration.<br />
<iframe width="755" height="500" frameborder="0" src="https://dl.dropbox.com/u/3824307/iGEM/youtube.html"></iframe><br />
</div><br />
</div> <br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/templates/wetnavTeam:Cornell/templates/wetnav2013-09-28T03:23:47Z<p>R.Lizarralde: re-added animation</p>
<hr />
<div><html class="no-js" lang="en"><br />
<body><br />
<div id="bodyContent"><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
<ul class="side-nav"><br />
<li><br />
<h6>Wet Lab</h6><br />
</li><br />
<li class="divider"></li><br />
<li id="liOverview"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li id="liChassis"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a> <br />
</li><br />
<li id="liFungalToolkit"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit">Fungal Toolkit</a><br />
<ul><br />
<li id="liRegulatoryElements"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/regulatory_elements">Regulatory Elements</a><br />
</li><br />
<li id="liSelectableMarkers"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/selectable_markers">Selectable Markers</a><br />
</li><br />
<li id="liCharacterization"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/characterization">Characterization</a><br />
</li><br />
<li id="liHomologousConstructs"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/homologous_constructs">Homologous Constructs</a><br />
</li><br />
<li id="liBioSafety"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">Biosafety</a><br />
</li><br />
<li id="liProtoplasting"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/protoplasting">Protoplasting</a> <br />
</li><br />
</ul><br />
</li><br />
<li id="liCarotenoids"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/carotenoids">Carotenoids</a><br />
</li><br />
<li id="liLimonene"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/limonene">Limonene</a><br />
</li><br />
<li id="liAntiFungals"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/antifungals">Antifungals</a><br />
</li><br />
<li id="liBioBricks"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/biobricks">BioBricks</a><br />
</li><br />
<li id="liFutureWork"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
<li id="liAnimation"><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/animation">Animation</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</body><br />
<script><br />
var h=window.location.href.toString()<br />
if(h == "https://2013.igem.org/Team:Cornell/project/wetlab")<br />
{ document.getElementById("liOverview").className="active"; }<br />
else if(h.indexOf("project/wetlab/chassis") != -1)<br />
{ document.getElementById("liChassis").className="active"; }<br />
else if(h.indexOf("project/wetlab/protoplasting") != -1)<br />
{ document.getElementById("liProtoplasting").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/regulatory_elements") != -1)<br />
{ document.getElementById("liRegulatoryElements").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/selectable_markers") != -1)<br />
{ document.getElementById("liSelectableMarkers").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/homologous_constructs") != -1)<br />
{ document.getElementById("liHomologousConstructs").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/characterization") != -1)<br />
{ document.getElementById("liCharacterization").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/biosafety") != -1)<br />
{ document.getElementById("liBioSafety").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/carotenoids") != -1)<br />
{ document.getElementById("liCarotenoids").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/limonene") != -1)<br />
{ document.getElementById("liLimonene").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit/antifungals") != -1)<br />
{ document.getElementById("liAntiFungals").className="active"; }<br />
else if(h.indexOf("project/wetlab/biobricks") != -1)<br />
{ document.getElementById("liBioBricks").className="active"; }<br />
else if(h.indexOf("project/wetlab/fungal_toolkit") != -1)<br />
{ var liFT = document.getElementById("liFungalToolkit")<br />
liFT.className="active";<br />
var childLi = liFT.children[1].children;<br />
for(var i = 0; i < childLi.length; i++) { childLi[i].className="inactive"; }<br />
}<br />
else if(h.indexOf("project/wetlab/animation") != -1)<br />
{ document.getElementById("liAnimation").className="active"; }<br />
else if(h.indexOf("roject/wetlab/future_work") != -1)<br />
{ document.getElementById("liFutureWork").className="active"; }<br />
</script><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/stylesheets/foundationTeam:Cornell/stylesheets/foundation2013-09-28T03:09:19Z<p>R.Lizarralde: </p>
<hr />
<div>.c-1, .c-2, .c-3, .c-4, .c-5, .c-6, .c-7, .c-8, .c-9, .c-10, .c-11, .c-12 { float: left; }<br />
<br />
.c-1, .c-2, .c-3, .c-4, .c-5, .c-6, .c-7, .c-8, .c-9, .c-10, .c-11, .c-12 { position: relative; min-height: 1px; padding: 0 15px; }<br />
<br />
.c-1 { width: 8.33333%; }<br />
<br />
.c-2 { width: 16.66667%; }<br />
<br />
.c-3 { width: 25%; }<br />
<br />
.c-4 { width: 33.33333%; }<br />
<br />
.c-5 { width: 41.66667%; }<br />
<br />
.c-6 { width: 50%; }<br />
<br />
.c-7 { width: 58.33333%; }<br />
<br />
.c-8 { width: 66.66667%; }<br />
<br />
.c-9 { width: 75%; }<br />
<br />
.c-10 { width: 83.33333%; }<br />
<br />
.c-11 { width: 91.66667%; }<br />
<br />
.c-12 { width: 100%; }<br />
<br />
/* Requires: normalize.css */<br />
/* Global Reset & Standards ---------------------- */<br />
* { -webkit-box-sizing: border-box; -moz-box-sizing: border-box; box-sizing: border-box; }<br />
<br />
html { font-size: 62.5%; }<br />
<br />
body { background: white; font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-size: 14px; line-height: 1; color: #222222; position: relative; -webkit-font-smoothing: antialiased; }<br />
<br />
/* Links ---------------------- */<br />
a { color: #2ba6cb; text-decoration: none; line-height: inherit; }<br />
<br />
a:hover { color: #2795b6; }<br />
<br />
a:focus { color: #2ba6cb; outline: none; }<br />
<br />
p a, p a:visited { line-height: inherit; }<br />
<br />
/* Misc ---------------------- */<br />
.left { float: left; }<br />
<br />
.right { float: right; }<br />
<br />
.text-left { text-align: left; }<br />
<br />
.text-right { text-align: right; }<br />
<br />
.text-center { text-align: center; }<br />
<br />
.hide { display: none; }<br />
<br />
.highlight { background: #ffff99; }<br />
<br />
#googlemap img, object, embed { max-width: none; }<br />
<br />
#map_canvas embed { max-width: none; }<br />
<br />
#map_canvas img { max-width: none; }<br />
<br />
#map_canvas object { max-width: none; }<br />
<br />
/* Reset for strange margins by default on <figure> elements */<br />
figure { margin: 0; }<br />
<br />
/* Base Type Styles Using Modular Scale ---------------------- */<br />
body, div, dl, dt, dd, ul, ol, li, h1, h2, h3, h4, h5, h6, pre, form, fieldset, p, blockquote, th, td { margin: 0; padding: 0; font-size: 14px; }<br />
<br />
p { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-weight: normal; font-size: 14px; line-height: 1.6; margin-bottom: 17px; }<br />
p.lead { font-size: 17.5px; line-height: 1.6; margin-bottom: 17px; }<br />
<br />
aside p { font-size: 13px; line-height: 1.35; font-style: italic; }<br />
<br />
h1, h2, h3, h4, h5, h6 { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; text-rendering: optimizeLegibility; line-height: 1.1; margin-bottom: 14px; margin-top: 14px; }<br />
h1 small, h2 small, h3 small, h4 small, h5 small, h6 small { font-size: 60%; color: #6f6f6f; line-height: 0; }<br />
<br />
h1 { font-size: 44px; }<br />
<br />
h2 { font-size: 37px; }<br />
<br />
h3 { font-size: 27px; }<br />
<br />
h4 { font-size: 23px; }<br />
<br />
h5 { font-size: 17px; }<br />
<br />
h6 { font-size: 14px; }<br />
<br />
hr { border: solid #ddd; border-width: 1px 0 0; clear: both; margin: 22px 0 21px; height: 0; }<br />
<br />
.subheader { line-height: 1.3; color: #6f6f6f; font-weight: 300; margin-bottom: 17px; }<br />
<br />
em, i { font-style: italic; line-height: inherit; }<br />
<br />
strong, b { font-weight: bold; line-height: inherit; }<br />
<br />
small { font-size: 60%; line-height: inherit; }<br />
<br />
code { font-weight: bold; background: #ffff99; }<br />
<br />
/* Lists ---------------------- */<br />
ul, ol { font-size: 14px; line-height: 1.6; margin-bottom: 17px; list-style-position: inside; }<br />
<br />
ul li ul, ul li ol { margin-left: 20px; margin-bottom: 0; }<br />
ul.square, ul.circle, ul.disc { margin-left: 17px; }<br />
ul.square { list-style-type: square; }<br />
ul.square li ul { list-style: inherit; }<br />
ul.circle { list-style-type: circle; }<br />
ul.circle li ul { list-style: inherit; }<br />
ul.disc { list-style-type: disc; }<br />
ul.disc li ul { list-style: inherit; }<br />
ul.no-bullet { list-style: none; }<br />
ul.large li { line-height: 21px; }<br />
<br />
ol li ul, ol li ol { margin-left: 20px; margin-bottom: 0; }<br />
<br />
/* Blockquotes ---------------------- */<br />
blockquote, blockquote p { line-height: 1.5; color: #6f6f6f; }<br />
<br />
blockquote { margin: 0 0 17px; padding: 9px 20px 0 19px; border-left: 1px solid #ddd; }<br />
blockquote cite { display: block; font-size: 13px; color: #555555; }<br />
blockquote cite:before { content: "\2014 \0020"; }<br />
blockquote cite a, blockquote cite a:visited { color: #555555; }<br />
<br />
abbr, acronym { text-transform: uppercase; font-size: 90%; color: #222222; border-bottom: 1px solid #ddd; cursor: help; }<br />
<br />
abbr { text-transform: none; }<br />
<br />
/* Print styles. Inlined to avoid required HTTP connection: www.phpied.com/delay-loading-your-print-css/ Credit to Paul Irish and HTML5 Boilerplate (html5boilerplate.com)<br />
*/<br />
.print-only { display: none !important; }<br />
<br />
@media print { * { background: transparent !important; color: black !important; box-shadow: none !important; text-shadow: none !important; filter: none !important; -ms-filter: none !important; }<br />
/* Black prints faster: h5bp.com/s */<br />
a, a:visited { text-decoration: underline; }<br />
a[href]:after { content: " (" attr(href) ")"; }<br />
abbr[title]:after { content: " (" attr(title) ")"; }<br />
.ir a:after, a[href^="javascript:"]:after, a[href^="#"]:after { content: ""; }<br />
/* Don't show links for images, or javascript/internal links */<br />
pre, blockquote { border: 1px solid #999; page-break-inside: avoid; }<br />
thead { display: table-header-group; }<br />
/* h5bp.com/t */<br />
tr, img { page-break-inside: avoid; }<br />
img { max-width: 100% !important; }<br />
@page { margin: 0.5cm; }<br />
p, h2, h3 { orphans: 3; widows: 3; }<br />
h2, h3 { page-break-after: avoid; }<br />
.hide-on-print { display: none !important; }<br />
.print-only { display: block !important; } }<br />
/* Requires globals.css */<br />
/* Standard Forms ---------------------- */<br />
form { margin: 0 0 19.41641px; }<br />
<br />
.row form .row { margin: 0 -6px; }<br />
.row form .row .column, .row form .row .columns { padding: 0 6px; }<br />
.row form .row.collapse { margin: 0; }<br />
.row form .row.collapse .column, .row form .row.collapse .columns { padding: 0; }<br />
<br />
label { font-size: 14px; color: #4d4d4d; cursor: pointer; display: block; font-weight: 500; margin-bottom: 3px; }<br />
label.right { float: none; text-align: right; }<br />
label.inline { line-height: 32px; margin: 0 0 12px 0; }<br />
<br />
@media only screen and (max-width: 767px) { label.right { text-align: left; } }<br />
.prefix, .postfix { display: block; position: relative; z-index: 2; text-align: center; width: 100%; padding-top: 0; padding-bottom: 0; height: 32px; line-height: 31px; }<br />
<br />
a.button.prefix, a.button.postfix { padding-left: 0; padding-right: 0; text-align: center; }<br />
<br />
span.prefix, span.postfix { background: #f2f2f2; border: 1px solid #cccccc; }<br />
<br />
.prefix { left: 2px; -moz-border-radius-topleft: 2px; -webkit-border-top-left-radius: 2px; border-top-left-radius: 2px; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; }<br />
<br />
.postfix { right: 2px; -moz-border-radius-topright: 2px; -webkit-border-top-right-radius: 2px; border-top-right-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
input[type="text"], input[type="password"], input[type="date"], input[type="datetime"], input[type="email"], input[type="number"], input[type="search"], input[type="tel"], input[type="time"], input[type="url"], textarea { border: 1px solid #cccccc; -webkit-border-radius: 2px; -moz-border-radius: 2px; -ms-border-radius: 2px; -o-border-radius: 2px; border-radius: 2px; -webkit-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); -moz-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); color: rgba(0, 0, 0, 0.75); display: block; font-size: 14px; margin: 0 0 12px 0; padding: 6px; height: 32px; width: 100%; -webkit-transition: all 0.15s linear; -moz-transition: all 0.15s linear; -o-transition: all 0.15s linear; transition: all 0.15s linear; }<br />
input[type="text"].oversize, input[type="password"].oversize, input[type="date"].oversize, input[type="datetime"].oversize, input[type="email"].oversize, input[type="number"].oversize, input[type="search"].oversize, input[type="tel"].oversize, input[type="time"].oversize, input[type="url"].oversize, textarea.oversize { font-size: 17px; padding: 4px 6px; }<br />
input[type="text"]:focus, input[type="password"]:focus, input[type="date"]:focus, input[type="datetime"]:focus, input[type="email"]:focus, input[type="number"]:focus, input[type="search"]:focus, input[type="tel"]:focus, input[type="time"]:focus, input[type="url"]:focus, textarea:focus { background: #fafafa; outline: none !important; border-color: #b3b3b3; }<br />
input[type="text"][disabled], input[type="password"][disabled], input[type="date"][disabled], input[type="datetime"][disabled], input[type="email"][disabled], input[type="number"][disabled], input[type="search"][disabled], input[type="tel"][disabled], input[type="time"][disabled], input[type="url"][disabled], textarea[disabled] { background-color: #ddd; }<br />
<br />
textarea { height: auto; }<br />
<br />
select { width: 100%; }<br />
<br />
/* Fieldsets */<br />
fieldset { border: solid 1px #ddd; border-radius: 3px; -webkit-border-radius: 3px; -moz-border-radius: 3px; padding: 12px 12px 0; margin: 18px 0; }<br />
fieldset legend { font-weight: bold; background: white; padding: 0 3px; margin: 0 0 0 -3px; }<br />
<br />
/* Errors */<br />
.error input, input.error, .error textarea, textarea.error { border-color: #c60f13; background-color: rgba(198, 15, 19, 0.1); }<br />
<br />
.error label, label.error { color: #c60f13; }<br />
<br />
.error small, small.error { display: block; padding: 6px 4px; margin-top: -13px; margin-bottom: 12px; background: #c60f13; color: #fff; font-size: 12px; font-size: 1.2rem; font-weight: bold; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
@media only screen and (max-width: 767px) { input[type="text"].one, input[type="password"].one, input[type="date"].one, input[type="datetime"].one, input[type="email"].one, input[type="number"].one, input[type="search"].one, input[type="tel"].one, input[type="time"].one, input[type="url"].one, textarea.one, .row textarea.one { width: 100% !important; }<br />
input[type="text"].two, .row input[type="text"].two, input[type="password"].two, .row input[type="password"].two, input[type="date"].two, .row input[type="date"].two, input[type="datetime"].two, .row input[type="datetime"].two, input[type="email"].two, .row input[type="email"].two, input[type="number"].two, .row input[type="number"].two, input[type="search"].two, .row input[type="search"].two, input[type="tel"].two, .row input[type="tel"].two, input[type="time"].two, .row input[type="time"].two, input[type="url"].two, .row input[type="url"].two, textarea.two, .row textarea.two { width: 100% !important; }<br />
input[type="text"].three, .row input[type="text"].three, input[type="password"].three, .row input[type="password"].three, input[type="date"].three, .row input[type="date"].three, input[type="datetime"].three, .row input[type="datetime"].three, input[type="email"].three, .row input[type="email"].three, input[type="number"].three, .row input[type="number"].three, input[type="search"].three, .row input[type="search"].three, input[type="tel"].three, .row input[type="tel"].three, input[type="time"].three, .row input[type="time"].three, input[type="url"].three, .row input[type="url"].three, textarea.three, .row textarea.three { width: 100% !important; }<br />
input[type="text"].four, .row input[type="text"].four, input[type="password"].four, .row input[type="password"].four, input[type="date"].four, .row input[type="date"].four, input[type="datetime"].four, .row input[type="datetime"].four, input[type="email"].four, .row input[type="email"].four, input[type="number"].four, .row input[type="number"].four, input[type="search"].four, .row input[type="search"].four, input[type="tel"].four, .row input[type="tel"].four, input[type="time"].four, .row input[type="time"].four, input[type="url"].four, .row input[type="url"].four, textarea.four, .row textarea.four { width: 100% !important; }<br />
input[type="text"].five, .row input[type="text"].five, input[type="password"].five, .row input[type="password"].five, input[type="date"].five, .row input[type="date"].five, input[type="datetime"].five, .row input[type="datetime"].five, input[type="email"].five, .row input[type="email"].five, input[type="number"].five, .row input[type="number"].five, input[type="search"].five, .row input[type="search"].five, input[type="tel"].five, .row input[type="tel"].five, input[type="time"].five, .row input[type="time"].five, input[type="url"].five, .row input[type="url"].five, textarea.five, .row textarea.five { width: 100% !important; }<br />
input[type="text"].six, .row input[type="text"].six, input[type="password"].six, .row input[type="password"].six, input[type="date"].six, .row input[type="date"].six, input[type="datetime"].six, .row input[type="datetime"].six, input[type="email"].six, .row input[type="email"].six, input[type="number"].six, .row input[type="number"].six, input[type="search"].six, .row input[type="search"].six, input[type="tel"].six, .row input[type="tel"].six, input[type="time"].six, .row input[type="time"].six, input[type="url"].six, .row input[type="url"].six, textarea.six, .row textarea.six { width: 100% !important; }<br />
input[type="text"].seven, .row input[type="text"].seven, input[type="password"].seven, .row input[type="password"].seven, input[type="date"].seven, .row input[type="date"].seven, input[type="datetime"].seven, .row input[type="datetime"].seven, input[type="email"].seven, .row input[type="email"].seven, input[type="number"].seven, .row input[type="number"].seven, input[type="search"].seven, .row input[type="search"].seven, input[type="tel"].seven, .row input[type="tel"].seven, input[type="time"].seven, .row input[type="time"].seven, input[type="url"].seven, .row input[type="url"].seven, textarea.seven, .row textarea.seven { width: 100% !important; }<br />
input[type="text"].eight, .row input[type="text"].eight, input[type="password"].eight, .row input[type="password"].eight, input[type="date"].eight, .row input[type="date"].eight, input[type="datetime"].eight, .row input[type="datetime"].eight, input[type="email"].eight, .row input[type="email"].eight, input[type="number"].eight, .row input[type="number"].eight, input[type="search"].eight, .row input[type="search"].eight, input[type="tel"].eight, .row input[type="tel"].eight, input[type="time"].eight, .row input[type="time"].eight, input[type="url"].eight, .row input[type="url"].eight, textarea.eight, .row textarea.eight { width: 100% !important; }<br />
input[type="text"].nine, .row input[type="text"].nine, input[type="password"].nine, .row input[type="password"].nine, input[type="date"].nine, .row input[type="date"].nine, input[type="datetime"].nine, .row input[type="datetime"].nine, input[type="email"].nine, .row input[type="email"].nine, input[type="number"].nine, .row input[type="number"].nine, input[type="search"].nine, .row input[type="search"].nine, input[type="tel"].nine, .row input[type="tel"].nine, input[type="time"].nine, .row input[type="time"].nine, input[type="url"].nine, .row input[type="url"].nine, textarea.nine, .row textarea.nine { width: 100% !important; }<br />
input[type="text"].ten, .row input[type="text"].ten, input[type="password"].ten, .row input[type="password"].ten, input[type="date"].ten, .row input[type="date"].ten, input[type="datetime"].ten, .row input[type="datetime"].ten, input[type="email"].ten, .row input[type="email"].ten, input[type="number"].ten, .row input[type="number"].ten, input[type="search"].ten, .row input[type="search"].ten, input[type="tel"].ten, .row input[type="tel"].ten, input[type="time"].ten, .row input[type="time"].ten, input[type="url"].ten, .row input[type="url"].ten, textarea.ten, .row textarea.ten { width: 100% !important; }<br />
input[type="text"].eleven, .row input[type="text"].eleven, input[type="password"].eleven, .row input[type="password"].eleven, input[type="date"].eleven, .row input[type="date"].eleven, input[type="datetime"].eleven, .row input[type="datetime"].eleven, input[type="email"].eleven, .row input[type="email"].eleven, input[type="number"].eleven, .row input[type="number"].eleven, input[type="search"].eleven, .row input[type="search"].eleven, input[type="tel"].eleven, .row input[type="tel"].eleven, input[type="time"].eleven, .row input[type="time"].eleven, input[type="url"].eleven, .row input[type="url"].eleven, textarea.eleven, .row textarea.eleven { width: 100% !important; }<br />
input[type="text"].twelve, .row input[type="text"].twelve, input[type="password"].twelve, .row input[type="password"].twelve, input[type="date"].twelve, .row input[type="date"].twelve, input[type="datetime"].twelve, .row input[type="datetime"].twelve, input[type="email"].twelve, .row input[type="email"].twelve, input[type="number"].twelve, .row input[type="number"].twelve, input[type="search"].twelve, .row input[type="search"].twelve, input[type="tel"].twelve, .row input[type="tel"].twelve, input[type="time"].twelve, .row input[type="time"].twelve, input[type="url"].twelve, .row input[type="url"].twelve, textarea.twelve, .row textarea.twelve { width: 100% !important; } }<br />
/* Custom Forms ---------------------- */<br />
form.custom { /* Custom input, disabled */ }<br />
form.custom span.custom { display: inline-block; width: 16px; height: 16px; position: relative; top: 2px; border: solid 1px #ccc; background: #fff; }<br />
form.custom span.custom.radio { -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; }<br />
form.custom span.custom.checkbox:before { content: ""; display: block; line-height: 0.8; height: 14px; width: 14px; text-align: center; position: absolute; top: 0; left: 0; font-size: 14px; color: #fff; }<br />
form.custom span.custom.radio.checked:before { content: ""; display: block; width: 8px; height: 8px; -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; background: #222; position: relative; top: 3px; left: 3px; }<br />
form.custom span.custom.checkbox.checked:before { content: "\00d7"; color: #222; }<br />
form.custom div.custom.dropdown { display: block; position: relative; width: auto; height: 28px; margin-bottom: 9px; margin-top: 2px; }<br />
form.custom div.custom.dropdown a.current { display: block; width: auto; line-height: 26px; min-height: 28px; padding: 0 38px 0 6px; border: solid 1px #ddd; color: #141414; background-color: #fff; white-space: nowrap; }<br />
form.custom div.custom.dropdown a.selector { position: absolute; width: 27px; height: 28px; display: block; right: 0; top: 0; border: solid 1px #ddd; }<br />
form.custom div.custom.dropdown a.selector:after { content: ""; display: block; content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #aaaaaa transparent transparent transparent; position: absolute; left: 50%; top: 50%; margin-top: -2px; margin-left: -5px; }<br />
form.custom div.custom.dropdown:hover a.selector:after, form.custom div.custom.dropdown.open a.selector:after { content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #222222 transparent transparent transparent; }<br />
form.custom div.custom.dropdown.open ul { display: block; z-index: 10; }<br />
form.custom div.custom.dropdown.small { width: 134px !important; }<br />
form.custom div.custom.dropdown.medium { width: 254px !important; }<br />
form.custom div.custom.dropdown.large { width: 434px !important; }<br />
form.custom div.custom.dropdown.expand { width: 100% !important; }<br />
form.custom div.custom.dropdown.open.small ul { width: 134px !important; }<br />
form.custom div.custom.dropdown.open.medium ul { width: 254px !important; }<br />
form.custom div.custom.dropdown.open.large ul { width: 434px !important; }<br />
form.custom div.custom.dropdown.open.expand ul { width: 100% !important; }<br />
form.custom div.custom.dropdown ul { position: absolute; width: auto; display: none; margin: 0; left: 0; top: 27px; margin: 0; padding: 0; background: #fff; background: rgba(255, 255, 255, 0.95); border: solid 1px #cccccc; }<br />
form.custom div.custom.dropdown ul li { color: #555; font-size: 13px; cursor: pointer; padding: 3px 38px 3px 6px; min-height: 18px; line-height: 18px; margin: 0; white-space: nowrap; list-style: none; }<br />
form.custom div.custom.dropdown ul li.selected { background: #cdebf5; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:after { content: "\2013"; position: absolute; right: 10px; }<br />
form.custom div.custom.dropdown ul li:hover { background-color: #e3f4f9; color: #222; }<br />
form.custom div.custom.dropdown ul li:hover:after { content: "\2013"; position: absolute; right: 10px; color: #8ed3e7; }<br />
form.custom div.custom.dropdown ul li.selected:hover { background: #cdebf5; cursor: default; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:hover:after { color: #000; }<br />
form.custom div.custom.dropdown ul.show { display: block; }<br />
form.custom .custom.disabled { background-color: #ddd; }<br />
<br />
/* Correct FF custom dropdown height */<br />
@-moz-document url-prefix() { form.custom div.custom.dropdown a.selector { height: 30px; } }<br />
<br />
.lt-ie9 form.custom div.custom.dropdown a.selector { height: 30px; }<br />
<br />
/* Requires globals.css */<br />
/* Standard Forms ---------------------- */<br />
form { margin: 0 0 19.41641px; }<br />
<br />
.row form .row { margin: 0 -6px; }<br />
.row form .row .column, .row form .row .columns { padding: 0 6px; }<br />
.row form .row.collapse { margin: 0; }<br />
.row form .row.collapse .column, .row form .row.collapse .columns { padding: 0; }<br />
<br />
label { font-size: 14px; color: #4d4d4d; cursor: pointer; display: block; font-weight: 500; margin-bottom: 3px; }<br />
label.right { float: none; text-align: right; }<br />
label.inline { line-height: 32px; margin: 0 0 12px 0; }<br />
<br />
@media only screen and (max-width: 767px) { label.right { text-align: left; } }<br />
.prefix, .postfix { display: block; position: relative; z-index: 2; text-align: center; width: 100%; padding-top: 0; padding-bottom: 0; height: 32px; line-height: 31px; }<br />
<br />
a.button.prefix, a.button.postfix { padding-left: 0; padding-right: 0; text-align: center; }<br />
<br />
span.prefix, span.postfix { background: #f2f2f2; border: 1px solid #cccccc; }<br />
<br />
.prefix { left: 2px; -moz-border-radius-topleft: 2px; -webkit-border-top-left-radius: 2px; border-top-left-radius: 2px; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; }<br />
<br />
.postfix { right: 2px; -moz-border-radius-topright: 2px; -webkit-border-top-right-radius: 2px; border-top-right-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
input[type="text"], input[type="password"], input[type="date"], input[type="datetime"], input[type="email"], input[type="number"], input[type="search"], input[type="tel"], input[type="time"], input[type="url"], textarea { border: 1px solid #cccccc; -webkit-border-radius: 2px; -moz-border-radius: 2px; -ms-border-radius: 2px; -o-border-radius: 2px; border-radius: 2px; -webkit-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); -moz-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); color: rgba(0, 0, 0, 0.75); display: block; font-size: 14px; margin: 0 0 12px 0; padding: 6px; height: 32px; width: 100%; -webkit-transition: all 0.15s linear; -moz-transition: all 0.15s linear; -o-transition: all 0.15s linear; transition: all 0.15s linear; }<br />
input[type="text"].oversize, input[type="password"].oversize, input[type="date"].oversize, input[type="datetime"].oversize, input[type="email"].oversize, input[type="number"].oversize, input[type="search"].oversize, input[type="tel"].oversize, input[type="time"].oversize, input[type="url"].oversize, textarea.oversize { font-size: 17px; padding: 4px 6px; }<br />
input[type="text"]:focus, input[type="password"]:focus, input[type="date"]:focus, input[type="datetime"]:focus, input[type="email"]:focus, input[type="number"]:focus, input[type="search"]:focus, input[type="tel"]:focus, input[type="time"]:focus, input[type="url"]:focus, textarea:focus { background: #fafafa; outline: none !important; border-color: #b3b3b3; }<br />
input[type="text"][disabled], input[type="password"][disabled], input[type="date"][disabled], input[type="datetime"][disabled], input[type="email"][disabled], input[type="number"][disabled], input[type="search"][disabled], input[type="tel"][disabled], input[type="time"][disabled], input[type="url"][disabled], textarea[disabled] { background-color: #ddd; }<br />
<br />
textarea { height: auto; }<br />
<br />
select { width: 100%; }<br />
<br />
/* Fieldsets */<br />
fieldset { border: solid 1px #ddd; border-radius: 3px; -webkit-border-radius: 3px; -moz-border-radius: 3px; padding: 12px 12px 0; margin: 18px 0; }<br />
fieldset legend { font-weight: bold; background: white; padding: 0 3px; margin: 0 0 0 -3px; }<br />
<br />
/* Errors */<br />
.error input, input.error, .error textarea, textarea.error { border-color: #c60f13; background-color: rgba(198, 15, 19, 0.1); }<br />
<br />
.error label, label.error { color: #c60f13; }<br />
<br />
.error small, small.error { display: block; padding: 6px 4px; margin-top: -13px; margin-bottom: 12px; background: #c60f13; color: #fff; font-size: 12px; font-size: 1.2rem; font-weight: bold; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
@media only screen and (max-width: 767px) { input[type="text"].one, .row input[type="text"].one, input[type="password"].one, .row input[type="password"].one, input[type="date"].one, .row input[type="date"].one, input[type="datetime"].one, .row input[type="datetime"].one, input[type="email"].one, .row input[type="email"].one, input[type="number"].one, .row input[type="number"].one, input[type="search"].one, .row input[type="search"].one, input[type="tel"].one, .row input[type="tel"].one, input[type="time"].one, .row input[type="time"].one, input[type="url"].one, .row input[type="url"].one, textarea.one, .row textarea.one { width: 100% !important; }<br />
input[type="text"].two, .row input[type="text"].two, input[type="password"].two, .row input[type="password"].two, input[type="date"].two, .row input[type="date"].two, input[type="datetime"].two, .row input[type="datetime"].two, input[type="email"].two, .row input[type="email"].two, input[type="number"].two, .row input[type="number"].two, input[type="search"].two, .row input[type="search"].two, input[type="tel"].two, .row input[type="tel"].two, input[type="time"].two, .row input[type="time"].two, input[type="url"].two, .row input[type="url"].two, textarea.two, .row textarea.two { width: 100% !important; }<br />
input[type="text"].three, .row input[type="text"].three, input[type="password"].three, .row input[type="password"].three, input[type="date"].three, .row input[type="date"].three, input[type="datetime"].three, .row input[type="datetime"].three, input[type="email"].three, .row input[type="email"].three, input[type="number"].three, .row input[type="number"].three, input[type="search"].three, .row input[type="search"].three, input[type="tel"].three, .row input[type="tel"].three, input[type="time"].three, .row input[type="time"].three, input[type="url"].three, .row input[type="url"].three, textarea.three, .row textarea.three { width: 100% !important; }<br />
input[type="text"].four, .row input[type="text"].four, input[type="password"].four, .row input[type="password"].four, input[type="date"].four, .row input[type="date"].four, input[type="datetime"].four, .row input[type="datetime"].four, input[type="email"].four, .row input[type="email"].four, input[type="number"].four, .row input[type="number"].four, input[type="search"].four, .row input[type="search"].four, input[type="tel"].four, .row input[type="tel"].four, input[type="time"].four, .row input[type="time"].four, input[type="url"].four, .row input[type="url"].four, textarea.four, .row textarea.four { width: 100% !important; }<br />
input[type="text"].five, .row input[type="text"].five, input[type="password"].five, .row input[type="password"].five, input[type="date"].five, .row input[type="date"].five, input[type="datetime"].five, .row input[type="datetime"].five, input[type="email"].five, .row input[type="email"].five, input[type="number"].five, .row input[type="number"].five, input[type="search"].five, .row input[type="search"].five, input[type="tel"].five, .row input[type="tel"].five, input[type="time"].five, .row input[type="time"].five, input[type="url"].five, .row input[type="url"].five, textarea.five, .row textarea.five { width: 100% !important; }<br />
input[type="text"].six, .row input[type="text"].six, input[type="password"].six, .row input[type="password"].six, input[type="date"].six, .row input[type="date"].six, input[type="datetime"].six, .row input[type="datetime"].six, input[type="email"].six, .row input[type="email"].six, input[type="number"].six, .row input[type="number"].six, input[type="search"].six, .row input[type="search"].six, input[type="tel"].six, .row input[type="tel"].six, input[type="time"].six, .row input[type="time"].six, input[type="url"].six, .row input[type="url"].six, textarea.six, .row textarea.six { width: 100% !important; }<br />
input[type="text"].seven, .row input[type="text"].seven, input[type="password"].seven, .row input[type="password"].seven, input[type="date"].seven, .row input[type="date"].seven, input[type="datetime"].seven, .row input[type="datetime"].seven, input[type="email"].seven, .row input[type="email"].seven, input[type="number"].seven, .row input[type="number"].seven, input[type="search"].seven, .row input[type="search"].seven, input[type="tel"].seven, .row input[type="tel"].seven, input[type="time"].seven, .row input[type="time"].seven, input[type="url"].seven, .row input[type="url"].seven, textarea.seven, .row textarea.seven { width: 100% !important; }<br />
input[type="text"].eight, .row input[type="text"].eight, input[type="password"].eight, .row input[type="password"].eight, input[type="date"].eight, .row input[type="date"].eight, input[type="datetime"].eight, .row input[type="datetime"].eight, input[type="email"].eight, .row input[type="email"].eight, input[type="number"].eight, .row input[type="number"].eight, input[type="search"].eight, .row input[type="search"].eight, input[type="tel"].eight, .row input[type="tel"].eight, input[type="time"].eight, .row input[type="time"].eight, input[type="url"].eight, .row input[type="url"].eight, textarea.eight, .row textarea.eight { width: 100% !important; }<br />
input[type="text"].nine, .row input[type="text"].nine, input[type="password"].nine, .row input[type="password"].nine, input[type="date"].nine, .row input[type="date"].nine, input[type="datetime"].nine, .row input[type="datetime"].nine, input[type="email"].nine, .row input[type="email"].nine, input[type="number"].nine, .row input[type="number"].nine, input[type="search"].nine, .row input[type="search"].nine, input[type="tel"].nine, .row input[type="tel"].nine, input[type="time"].nine, .row input[type="time"].nine, input[type="url"].nine, .row input[type="url"].nine, textarea.nine, .row textarea.nine { width: 100% !important; }<br />
input[type="text"].ten, .row input[type="text"].ten, input[type="password"].ten, .row input[type="password"].ten, input[type="date"].ten, .row input[type="date"].ten, input[type="datetime"].ten, .row input[type="datetime"].ten, input[type="email"].ten, .row input[type="email"].ten, input[type="number"].ten, .row input[type="number"].ten, input[type="search"].ten, .row input[type="search"].ten, input[type="tel"].ten, .row input[type="tel"].ten, input[type="time"].ten, .row input[type="time"].ten, input[type="url"].ten, .row input[type="url"].ten, textarea.ten, .row textarea.ten { width: 100% !important; }<br />
input[type="text"].eleven, .row input[type="text"].eleven, input[type="password"].eleven, .row input[type="password"].eleven, input[type="date"].eleven, .row input[type="date"].eleven, input[type="datetime"].eleven, .row input[type="datetime"].eleven, input[type="email"].eleven, .row input[type="email"].eleven, input[type="number"].eleven, .row input[type="number"].eleven, input[type="search"].eleven, .row input[type="search"].eleven, input[type="tel"].eleven, .row input[type="tel"].eleven, input[type="time"].eleven, .row input[type="time"].eleven, input[type="url"].eleven, .row input[type="url"].eleven, textarea.eleven, .row textarea.eleven { width: 100% !important; }<br />
input[type="text"].twelve, .row input[type="text"].twelve, input[type="password"].twelve, .row input[type="password"].twelve, input[type="date"].twelve, .row input[type="date"].twelve, input[type="datetime"].twelve, .row input[type="datetime"].twelve, input[type="email"].twelve, .row input[type="email"].twelve, input[type="number"].twelve, .row input[type="number"].twelve, input[type="search"].twelve, .row input[type="search"].twelve, input[type="tel"].twelve, .row input[type="tel"].twelve, input[type="time"].twelve, .row input[type="time"].twelve, input[type="url"].twelve, .row input[type="url"].twelve, textarea.twelve, .row textarea.twelve { width: 100% !important; } }<br />
/* Custom Forms ---------------------- */<br />
form.custom { /* Custom input, disabled */ }<br />
form.custom span.custom { display: inline-block; width: 16px; height: 16px; position: relative; top: 2px; border: solid 1px #ccc; background: #fff; }<br />
form.custom span.custom.radio { -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; }<br />
form.custom span.custom.checkbox:before { content: ""; display: block; line-height: 0.8; height: 14px; width: 14px; text-align: center; position: absolute; top: 0; left: 0; font-size: 14px; color: #fff; }<br />
form.custom span.custom.radio.checked:before { content: ""; display: block; width: 8px; height: 8px; -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; background: #222; position: relative; top: 3px; left: 3px; }<br />
form.custom span.custom.checkbox.checked:before { content: "\00d7"; color: #222; }<br />
form.custom div.custom.dropdown { display: block; position: relative; width: auto; height: 28px; margin-bottom: 9px; margin-top: 2px; }<br />
form.custom div.custom.dropdown a.current { display: block; width: auto; line-height: 26px; min-height: 28px; padding: 0 38px 0 6px; border: solid 1px #ddd; color: #141414; background-color: #fff; white-space: nowrap; }<br />
form.custom div.custom.dropdown a.selector { position: absolute; width: 27px; height: 28px; display: block; right: 0; top: 0; border: solid 1px #ddd; }<br />
form.custom div.custom.dropdown a.selector:after { content: ""; display: block; content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #aaaaaa transparent transparent transparent; position: absolute; left: 50%; top: 50%; margin-top: -2px; margin-left: -5px; }<br />
form.custom div.custom.dropdown:hover a.selector:after, form.custom div.custom.dropdown.open a.selector:after { content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #222222 transparent transparent transparent; }<br />
form.custom div.custom.dropdown.open ul { display: block; z-index: 10; }<br />
form.custom div.custom.dropdown.small { width: 134px !important; }<br />
form.custom div.custom.dropdown.medium { width: 254px !important; }<br />
form.custom div.custom.dropdown.large { width: 434px !important; }<br />
form.custom div.custom.dropdown.expand { width: 100% !important; }<br />
form.custom div.custom.dropdown.open.small ul { width: 134px !important; }<br />
form.custom div.custom.dropdown.open.medium ul { width: 254px !important; }<br />
form.custom div.custom.dropdown.open.large ul { width: 434px !important; }<br />
form.custom div.custom.dropdown.open.expand ul { width: 100% !important; }<br />
form.custom div.custom.dropdown ul { position: absolute; width: auto; display: none; margin: 0; left: 0; top: 27px; margin: 0; padding: 0; background: #fff; background: rgba(255, 255, 255, 0.95); border: solid 1px #cccccc; }<br />
form.custom div.custom.dropdown ul li { color: #555; font-size: 13px; cursor: pointer; padding: 3px 38px 3px 6px; min-height: 18px; line-height: 18px; margin: 0; white-space: nowrap; list-style: none; }<br />
form.custom div.custom.dropdown ul li.selected { background: #cdebf5; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:after { content: "\2013"; position: absolute; right: 10px; }<br />
form.custom div.custom.dropdown ul li:hover { background-color: #e3f4f9; color: #222; }<br />
form.custom div.custom.dropdown ul li:hover:after { content: "\2013"; position: absolute; right: 10px; color: #8ed3e7; }<br />
form.custom div.custom.dropdown ul li.selected:hover { background: #cdebf5; cursor: default; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:hover:after { color: #000; }<br />
form.custom div.custom.dropdown ul.show { display: block; }<br />
form.custom .custom.disabled { background-color: #ddd; }<br />
<br />
/* Correct FF custom dropdown height */<br />
@-moz-document url-prefix() { form.custom div.custom.dropdown a.selector { height: 30px; } }<br />
<br />
.lt-ie9 form.custom div.custom.dropdown a.selector { height: 30px; }<br />
<br />
/* Base Type Styles Using Modular Scale ---------------------- */<br />
body, div, dl, dt, dd, ul, ol, li, h1, h2, h3, h4, h5, h6, pre, form, fieldset, p, blockquote, th, td { margin: 0; padding: 0; font-size: 14px; }<br />
<br />
ul.orbit-bullets { margin-bottom: 17px; }<br />
<br />
p { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-weight: normal; font-size: 14px; line-height: 1.6; margin-bottom: 17px; }<br />
p.lead { font-size: 17.5px; line-height: 1.6; margin-bottom: 17px; }<br />
<br />
aside p { font-size: 13px; line-height: 1.35; font-style: italic; }<br />
<br />
h1, h2, h3, h4, h5, h6 { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; text-rendering: optimizeLegibility; line-height: 1.1; margin-bottom: 14px; margin-top: 14px; }<br />
h1 small, h2 small, h3 small, h4 small, h5 small, h6 small { font-size: 60%; color: #6f6f6f; line-height: 0; }<br />
<br />
h1 { font-size: 44px; }<br />
<br />
h2 { font-size: 37px; }<br />
<br />
h3 { font-size: 27px; }<br />
<br />
h4 { font-size: 23px; }<br />
<br />
h5 { font-size: 17px; }<br />
<br />
h6 { font-size: 14px; }<br />
<br />
hr { border: solid #ddd; border-width: 1px 0 0; clear: both; margin: 22px 0 21px; height: 0; }<br />
<br />
.subheader { line-height: 1.3; color: #6f6f6f; font-weight: 300; margin-bottom: 17px; }<br />
<br />
em, i { font-style: italic; line-height: inherit; }<br />
<br />
strong, b { font-weight: bold; line-height: inherit; }<br />
<br />
small { font-size: 60%; line-height: inherit; }<br />
<br />
code { font-weight: bold; background: #ffff99; }<br />
<br />
/* The Grid ---------------------- */<br />
.row { width: 940px; max-width: 100%; min-width: 768px; margin: 0 auto; }<br />
.row .row { width: auto; max-width: none; min-width: 0; margin: 0 -15px; }<br />
.row.collapse .column, .row.collapse .columns { padding: 0; }<br />
.row .row { width: auto; max-width: none; min-width: 0; margin: 0 -15px; }<br />
.row .row.collapse { margin: 0; }<br />
<br />
.column, .columns { float: left; min-height: 1px; padding: 0 15px; position: relative; }<br />
.column.centered, .columns.centered { float: none; margin: 0 auto; }<br />
<br />
[class*="column"] + [class*="column"]:last-child { float: right; }<br />
<br />
[class*="column"] + [class*="column"].end { float: left; }<br />
<br />
.one, .row .one { width: 8.33333%; }<br />
<br />
.two, .row .two { width: 16.66667%; }<br />
<br />
.three, .row .three { width: 25%; }<br />
<br />
.four, .row .four { width: 33.33333%; }<br />
<br />
.five, .row .five { width: 41.66667%; }<br />
<br />
.six, .row .six { width: 50%; }<br />
<br />
.seven, .row .seven { width: 58.33333%; }<br />
<br />
.eight, .row .eight { width: 66.66667%; }<br />
<br />
.nine, .row .nine { width: 75%; }<br />
<br />
.ten, .row .ten { width: 83.33333%; }<br />
<br />
.eleven, .row .eleven { width: 91.66667%; }<br />
<br />
.twelve, .row .twelve { width: 100%; }<br />
<br />
.row .offset-by-one { margin-left: 8.33333%; }<br />
<br />
.row .offset-by-two { margin-left: 16.66667%; }<br />
<br />
.row .offset-by-three { margin-left: 25%; }<br />
<br />
.row .offset-by-four { margin-left: 33.33333%; }<br />
<br />
.row .offset-by-five { margin-left: 41.66667%; }<br />
<br />
.row .offset-by-six { margin-left: 50%; }<br />
<br />
.row .offset-by-seven { margin-left: 58.33333%; }<br />
<br />
.row .offset-by-eight { margin-left: 66.66667%; }<br />
<br />
.row .offset-by-nine { margin-left: 75%; }<br />
<br />
.row .offset-by-ten { margin-left: 83.33333%; }<br />
<br />
.push-two { left: 16.66667%; }<br />
<br />
.pull-two { right: 16.66667%; }<br />
<br />
.push-three { left: 25%; }<br />
<br />
.pull-three { right: 25%; }<br />
<br />
.push-four { left: 33.33333%; }<br />
<br />
.pull-four { right: 33.33333%; }<br />
<br />
.push-five { left: 41.66667%; }<br />
<br />
.pull-five { right: 41.66667%; }<br />
<br />
.push-six { left: 50%; }<br />
<br />
.pull-six { right: 50%; }<br />
<br />
.push-seven { left: 58.33333%; }<br />
<br />
.pull-seven { right: 58.33333%; }<br />
<br />
.push-eight { left: 66.66667%; }<br />
<br />
.pull-eight { right: 66.66667%; }<br />
<br />
.push-nine { left: 75%; }<br />
<br />
.pull-nine { right: 75%; }<br />
<br />
.push-ten { left: 83.33333%; }<br />
<br />
.pull-ten { right: 83.33333%; }<br />
<br />
img, object, embed { max-width: 100%; height: auto; }<br />
<br />
object, embed { height: 100%; }<br />
<br />
img { -ms-interpolation-mode: bicubic; }<br />
<br />
#map_canvas img, .map_canvas img { max-width: none!important; }<br />
<br />
/* Nicolas Gallagher's micro clearfix */<br />
.row { *zoom: 1; }<br />
.row:before, .row:after { content: ""; display: table; }<br />
.row:after { clear: both; }<br />
<br />
/* Mobile Grid and Overrides ---------------------- */<br />
@media only screen and (max-width: 767px) { body { -webkit-text-size-adjust: none; -ms-text-size-adjust: none; width: 100%; min-width: 0; margin-left: 0; margin-right: 0; padding-left: 0; padding-right: 0; }<br />
.row { width: auto; min-width: 0; margin-left: 0; margin-right: 0; }<br />
.column, .columns { width: auto !important; float: none; }<br />
.column:last-child, .columns:last-child { float: none; }<br />
[class*="column"] + [class*="column"]:last-child { float: none; }<br />
.column:before, .columns:before, .column:after, .columns:after { content: ""; display: table; }<br />
.column:after, .columns:after { clear: both; }<br />
.offset-by-one, .offset-by-two, .offset-by-three, .offset-by-four, .offset-by-five, .offset-by-six, .offset-by-seven, .offset-by-eight, .offset-by-nine, .offset-by-ten { margin-left: 0 !important; }<br />
.push-two, .push-three, .push-four, .push-five, .push-six, .push-seven, .push-eight, .push-nine, .push-ten { left: auto; }<br />
.pull-two, .pull-three, .pull-four, .pull-five, .pull-six, .pull-seven, .pull-eight, .pull-nine, .pull-ten { right: auto; }<br />
/* Mobile 4-column Grid */<br />
.row .mobile-one { width: 25% !important; float: left; padding: 0 15px; }<br />
.row .mobile-one:last-child { float: right; }<br />
.row.collapse .mobile-one { padding: 0; }<br />
.row .mobile-two { width: 50% !important; float: left; padding: 0 15px; }<br />
.row .mobile-two:last-child { float: right; }<br />
.row.collapse .mobile-two { padding: 0; }<br />
.row .mobile-three { width: 75% !important; float: left; padding: 0 15px; }<br />
.row .mobile-three:last-child { float: right; }<br />
.row.collapse .mobile-three { padding: 0; }<br />
.row .mobile-four { width: 100% !important; float: left; padding: 0 15px; }<br />
.row .mobile-four:last-child { float: right; }<br />
.row.collapse .mobile-four { padding: 0; }<br />
.push-one-mobile { left: 25%; }<br />
.pull-one-mobile { right: 25%; }<br />
.push-two-mobile { left: 50%; }<br />
.pull-two-mobile { right: 50%; }<br />
.push-three-mobile { left: 75%; }<br />
.pull-three-mobile { right: 75%; } }<br />
/* Block Grids ---------------------- */<br />
/* These are 2-up, 3-up, 4-up and 5-up ULs, suited<br />
for repeating blocks of content. Add 'mobile' to<br />
them to switch them just like the layout grid<br />
(one item per line) on phones<br />
<br />
For IE7/8 compatibility block-grid items need to be<br />
the same height. You can optionally uncomment the<br />
lines below to support arbitrary height, but know<br />
that IE7/8 do not support :nth-child.<br />
-------------------------------------------------- */<br />
.block-grid { display: block; overflow: hidden; padding: 0; }<br />
.block-grid > li { display: block; height: auto; float: left; }<br />
.block-grid.one-up { margin: 0; }<br />
.block-grid.one-up > li { width: 100%; padding: 0 0 15px; }<br />
.block-grid.two-up { margin: 0 -15px; }<br />
.block-grid.two-up > li { width: 50%; padding: 0 15px 15px; }<br />
.block-grid.two-up > li:nth-child(2n+1) { clear: both; }<br />
.block-grid.three-up { margin: 0 -12px; }<br />
.block-grid.three-up > li { width: 33.33%; padding: 0 12px 12px; }<br />
.block-grid.three-up > li:nth-child(3n+1) { clear: both; }<br />
.block-grid.four-up { margin: 0 -10px; }<br />
.block-grid.four-up > li { width: 25%; padding: 0 10px 10px; }<br />
.block-grid.four-up > li:nth-child(4n+1) { clear: both; }<br />
.block-grid.five-up { margin: 0 -8px; }<br />
.block-grid.five-up > li { width: 20%; padding: 0 8px 8px; }<br />
.block-grid.five-up > li:nth-child(5n+1) { clear: both; }<br />
<br />
/* Mobile Block Grids */<br />
@media only screen and (max-width: 767px) { .block-grid.mobile > li { float: none; width: 100%; margin-left: 0; }<br />
.block-grid > li { clear: none !important; }<br />
.block-grid.mobile-two-up > li { width: 50%; }<br />
.block-grid.mobile-two-up > li:nth-child(2n+1) { clear: both; }<br />
.block-grid.mobile-three-up > li { width: 33.33%; }<br />
.block-grid.mobile-three-up > li:nth-child(3n+1) { clear: both !important; }<br />
.block-grid.mobile-four-up > li { width: 25%; }<br />
.block-grid.mobile-four-up > li:nth-child(4n+1) { clear: both; }<br />
.block-grid.mobile-five-up > li:nth-child(5n+1) { clear: both; } }<br />
/* Requires globals.css */<br />
/* Normal Buttons ---------------------- */<br />
.button { width: auto; background: #2ba6cb; border: 1px solid #1e728c; -webkit-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; color: white; cursor: pointer; display: inline-block; font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-size: 14px; font-weight: bold; line-height: 1; margin: 0; outline: none; padding: 10px 20px 11px; position: relative; text-align: center; text-decoration: none; -webkit-transition: background-color 0.15s ease-in-out; -moz-transition: background-color 0.15s ease-in-out; -o-transition: background-color 0.15s ease-in-out; transition: background-color 0.15s ease-in-out; /* Hovers */ /* Sizes */ /* Colors */ /* Radii */ /* Layout */ /* Disabled ---------- */ }<br />
.button:hover { color: white; background-color: #2284a1; }<br />
.button:active { -webkit-box-shadow: 0 1px 0 rgba(0, 0, 0, 0.2) inset; -moz-box-shadow: 0 1px 0 rgba(0, 0, 0, 0.2) inset; box-shadow: 0 1px 0 rgba(0, 0, 0, 0.2) inset; }<br />
.button:focus { -webkit-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; color: white; }<br />
.button.large { font-size: 17px; padding: 15px 30px 16px; }<br />
.button.medium { font-size: 14px; }<br />
.button.small { font-size: 11px; padding: 7px 14px 8px; }<br />
.button.tiny { font-size: 10px; padding: 5px 10px 6px; }<br />
.button.expand { width: 100%; text-align: center; }<br />
.button.primary { background-color: #2ba6cb; border: 1px solid #1e728c; }<br />
.button.primary:hover { background-color: #2284a1; }<br />
.button.primary:focus { -webkit-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.success { background-color: #5da423; border: 1px solid #396516; }<br />
.button.success:hover { background-color: #457a1a; }<br />
.button.success:focus { -webkit-box-shadow: 0 0 5px #5da423, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 5px #5da423, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 5px #5da423, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.alert { background-color: #c60f13; border: 1px solid #7f0a0c; }<br />
.button.alert:hover { background-color: #970b0e; }<br />
.button.alert:focus { -webkit-box-shadow: 0 0 4px #c60f13, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 4px #c60f13, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 4px #c60f13, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.secondary { background-color: #e9e9e9; color: #1d1d1d; border: 1px solid #c3c3c3; }<br />
.button.secondary:hover { background-color: #d0d0d0; }<br />
.button.secondary:focus { -webkit-box-shadow: 0 0 5px #e9e9e9, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 5px #e9e9e9, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 5px #e9e9e9, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.radius { -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
.button.round { -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; }<br />
.button.full-width { width: 100%; text-align: center; padding-left: 0px !important; padding-right: 0px !important; }<br />
.button.left-align { text-align: left; text-indent: 12px; }<br />
.button.disabled, .button[disabled] { opacity: 0.6; cursor: default; background: #2ba6cb; -webkit-box-shadow: none; -moz-box-shadow: none; box-shadow: none; }<br />
.button.disabled :hover, .button[disabled] :hover { background: #2ba6cb; }<br />
.button.disabled.success, .button[disabled].success { background-color: #5da423; }<br />
.button.disabled.success:hover, .button[disabled].success:hover { background-color: #5da423; }<br />
.button.disabled.alert, .button[disabled].alert { background-color: #c60f13; }<br />
.button.disabled.alert:hover, .button[disabled].alert:hover { background-color: #c60f13; }<br />
.button.disabled.secondary, .button[disabled].secondary { background-color: #e9e9e9; }<br />
.button.disabled.secondary:hover, .button[disabled].secondary:hover { background-color: #e9e9e9; }<br />
<br />
/* Don't use native buttons on iOS */<br />
input[type=submit].button, button.button { -webkit-appearance: none; }<br />
<br />
@media only screen and (max-width: 767px) { .button { display: block; }<br />
button.button, input[type="submit"].button { width: 100%; padding-left: 0; padding-right: 0; } }<br />
/* Correct FF button padding */<br />
@-moz-document url-prefix() { button::-moz-focus-inner, input[type="reset"]::-moz-focus-inner, input[type="button"]::-moz-focus-inner, input[type="submit"]::-moz-focus-inner, input[type="file"] > input[type="button"]::-moz-focus-inner { border: none; padding: 0; }<br />
input[type="submit"].tiny.button { padding: 3px 10px 4px; }<br />
input[type="submit"].small.button { padding: 5px 14px 6px; }<br />
input[type="submit"].button, input[type=submit].medium.button { padding: 8px 20px 9px; }<br />
input[type="submit"].large.button { padding: 13px 30px 14px; } }<br />
<br />
/* Buttons with Dropdowns ---------------------- */<br />
.button.dropdown { position: relative; padding-right: 44px; /* Sizes */ /* Triangles */ /* Flyout List */ /* Split Dropdown Buttons */ }<br />
.button.dropdown.large { padding-right: 60px; }<br />
.button.dropdown.small { padding-right: 28px; }<br />
.button.dropdown.tiny { padding-right: 20px; }<br />
.button.dropdown:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: white transparent transparent transparent; position: absolute; top: 50%; right: 20px; margin-top: -2px; }<br />
.button.dropdown.large:after { content: ""; display: block; width: 0; height: 0; border: solid 7px; border-color: white transparent transparent transparent; margin-top: -3px; right: 30px; }<br />
.button.dropdown.small:after { content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: white transparent transparent transparent; margin-top: -2px; right: 14px; }<br />
.button.dropdown.tiny:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: white transparent transparent transparent; margin-top: -1px; right: 10px; }<br />
.button.dropdown > ul { -webkit-box-sizing: content-box; -moz-box-sizing: content-box; box-sizing: content-box; display: none; position: absolute; left: -1px; background: #fff; background: rgba(255, 255, 255, 0.95); list-style: none; margin: 0; padding: 0; border: 1px solid #cccccc; border-top: none; min-width: 100%; z-index: 40; }<br />
.button.dropdown > ul li { width: 100%; cursor: pointer; padding: 0; min-height: 18px; line-height: 18px; margin: 0; white-space: nowrap; list-style: none; }<br />
.button.dropdown > ul li a { display: block; color: #555; font-size: 13px; font-weight: normal; padding: 6px 14px; text-align: left; }<br />
.button.dropdown > ul li:hover { background-color: #e3f4f9; color: #222; }<br />
.button.dropdown > ul li.divider { min-height: 0; padding: 0; height: 1px; margin: 4px 0; background: #ededed; }<br />
.button.dropdown.up > ul { border-top: 1px solid #cccccc; border-bottom: none; }<br />
.button.dropdown ul.no-hover.show-dropdown { display: block !important; }<br />
.button.dropdown:hover > ul.no-hover { display: none; }<br />
.button.dropdown.split { padding: 0; position: relative; /* Sizes */ /* Triangle Spans */ /* Colors */ }<br />
.button.dropdown.split:after { display: none; }<br />
.button.dropdown.split:hover { background-color: #2ba6cb; }<br />
.button.dropdown.split.alert:hover { background-color: #c60f13; }<br />
.button.dropdown.split.success:hover { background-color: #5da423; }<br />
.button.dropdown.split.secondary:hover { background-color: #e9e9e9; }<br />
.button.dropdown.split > a { color: white; display: block; padding: 10px 50px 11px 20px; -webkit-transition: background-color 0.15s ease-in-out; -moz-transition: background-color 0.15s ease-in-out; -o-transition: background-color 0.15s ease-in-out; transition: background-color 0.15s ease-in-out; }<br />
.button.dropdown.split > a:hover { background-color: #2284a1; }<br />
.button.dropdown.split.large > a { padding: 15px 75px 16px 30px; }<br />
.button.dropdown.split.small > a { padding: 7px 35px 8px 14px; }<br />
.button.dropdown.split.tiny > a { padding: 5px 25px 6px 10px; }<br />
.button.dropdown.split > span { background-color: #2ba6cb; position: absolute; right: 0; top: 0; height: 100%; width: 30px; border-left: 1px solid #1e728c; -webkit-box-shadow: 1px 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 1px 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 1px 1px 0 rgba(255, 255, 255, 0.5) inset; -webkit-transition: background-color 0.15s ease-in-out; -moz-transition: background-color 0.15s ease-in-out; -o-transition: background-color 0.15s ease-in-out; transition: background-color 0.15s ease-in-out; }<br />
.button.dropdown.split > span:hover { background-color: #2284a1; }<br />
.button.dropdown.split > span:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: white transparent transparent transparent; position: absolute; top: 50%; left: 50%; margin-left: -6px; margin-top: -2px; }<br />
.button.dropdown.split.secondary > span:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: #1d1d1d transparent transparent transparent; }<br />
.button.dropdown.split.large span { width: 45px; }<br />
.button.dropdown.split.small span { width: 21px; }<br />
.button.dropdown.split.tiny span { width: 15px; }<br />
.button.dropdown.split.large span:after { content: ""; display: block; width: 0; height: 0; border: solid 7px; border-color: white transparent transparent transparent; margin-top: -3px; margin-left: -7px; }<br />
.button.dropdown.split.small span:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: white transparent transparent transparent; margin-top: -1px; margin-left: -4px; }<br />
.button.dropdown.split.tiny span:after { content: ""; display: block; width: 0; height: 0; border: solid 3px; border-color: white transparent transparent transparent; margin-top: -1px; margin-left: -3px; }<br />
.button.dropdown.split.alert > span { background-color: #c60f13; border-left-color: #7f0a0c; }<br />
.button.dropdown.split.success > span { background-color: #5da423; border-left-color: #396516; }<br />
.button.dropdown.split.secondary > span { background-color: #e9e9e9; border-left-color: #c3c3c3; }<br />
.button.dropdown.split.secondary > a { color: #1d1d1d; }<br />
.button.dropdown.split.alert > a:hover, .button.dropdown.split.alert > span:hover { background-color: #970b0e; }<br />
.button.dropdown.split.success > a:hover, .button.dropdown.split.success > span:hover { background-color: #457a1a; }<br />
.button.dropdown.split.secondary > a:hover, .button.dropdown.split.secondary > span:hover { background-color: #d0d0d0; }<br />
<br />
/* Button Groups ---------------------- */<br />
ul.button-group { list-style: none; padding: 0; margin: 0 0 12px; *zoom: 1; }<br />
ul.button-group:before, ul.button-group:after { content: ""; display: table; }<br />
ul.button-group:after { clear: both; }<br />
ul.button-group li { padding: 0; margin: 0 0 0 -1px; float: left; }<br />
ul.button-group li:first-child { margin-left: 0; }<br />
ul.button-group.radius li a.button, ul.button-group.radius li a.button.radius, ul.button-group.radius li a.button-rounded { -webkit-border-radius: 0px; -moz-border-radius: 0px; -ms-border-radius: 0px; -o-border-radius: 0px; border-radius: 0px; }<br />
ul.button-group.radius li:first-child a.button, ul.button-group.radius li:first-child a.button.radius { -moz-border-radius-topleft: 3px; -webkit-border-top-left-radius: 3px; border-top-left-radius: 3px; -moz-border-radius-bottomleft: 3px; -webkit-border-bottom-left-radius: 3px; border-bottom-left-radius: 3px; }<br />
ul.button-group.radius li:first-child a.button.rounded { -moz-border-radius-topleft: 1000px; -webkit-border-top-left-radius: 1000px; border-top-left-radius: 1000px; -moz-border-radius-bottomleft: 1000px; -webkit-border-bottom-left-radius: 1000px; border-bottom-left-radius: 1000px; }<br />
ul.button-group.radius li:last-child a.button, ul.button-group.radius li:last-child a.button.radius { -moz-border-radius-topright: 3px; -webkit-border-top-right-radius: 3px; border-top-right-radius: 3px; -moz-border-radius-bottomright: 3px; -webkit-border-bottom-right-radius: 3px; border-bottom-right-radius: 3px; }<br />
ul.button-group.radius li:last-child a.button.rounded { -moz-border-radius-topright: 1000px; -webkit-border-top-right-radius: 1000px; border-top-right-radius: 1000px; -moz-border-radius-bottomright: 1000px; -webkit-border-bottom-right-radius: 1000px; border-bottom-right-radius: 1000px; }<br />
ul.button-group.even a.button { width: 100%; }<br />
ul.button-group.even.two-up li { width: 50%; }<br />
ul.button-group.even.three-up li { width: 33.3%; }<br />
ul.button-group.even.three-up li:first-child { width: 33.4%; }<br />
ul.button-group.even.four-up li { width: 25%; }<br />
ul.button-group.even.five-up li { width: 20%; }<br />
<br />
@media only screen and (max-width: 767px) { .button-group button.button, .button-group input[type="submit"].button { width: auto; padding: 10px 20px 11px; }<br />
.button-group button.button.large, .button-group input[type="submit"].button.large { padding: 15px 30px 16px; }<br />
.button-group button.button.medium, .button-group input[type="submit"].button.medium { padding: 10px 20px 11px; }<br />
.button-group button.button.small, .button-group input[type="submit"].button.small { padding: 7px 14px 8px; }<br />
.button-group button.button.tiny, .button-group input[type="submit"].button.tiny { padding: 5px 10px 6px; }<br />
.button-group.even button.button, .button-group.even input[type="submit"].button { width: 100%; padding-left: 0; padding-right: 0; } }<br />
div.button-bar { overflow: hidden; }<br />
div.button-bar ul.button-group { float: left; margin-right: 8px; }<br />
div.button-bar ul.button-group:last-child { margin-left: 0; }<br />
<br />
/* Requires globals.css */<br />
.nav-bar { height: 40px; background: #4d4d4d; margin-top: 20px; padding: 0; }<br />
.nav-bar > li { float: left; display: block; position: relative; padding: 0; margin: 0; border: 1px solid #333333; border-right: none; line-height: 38px; -webkit-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset; -moz-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset; box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset; }<br />
.nav-bar > li:first-child { -webkit-box-shadow: 0 0 0; -moz-box-shadow: 0 0 0; box-shadow: 0 0 0; }<br />
.nav-bar > li:last-child { border-right: solid 1px #333333; -webkit-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset, 1px 0 0 rgba(255, 255, 255, 0.2); -moz-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset, 1px 0 0 rgba(255, 255, 255, 0.2); box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset, 1px 0 0 rgba(255, 255, 255, 0.2); }<br />
.nav-bar > li.active { background: #2ba6cb; border-color: #2284a1; }<br />
.nav-bar > li.active > a { color: white; cursor: default; }<br />
.nav-bar > li.active:hover { background: #2ba6cb; cursor: default; }<br />
.nav-bar > li:hover { background: #333333; }<br />
.nav-bar > li > a { color: #e6e6e6; }<br />
.nav-bar > li ul { margin-bottom: 0; }<br />
.nav-bar > li .flyout { display: none; }<br />
.nav-bar > li.has-flyout > a:first-child { padding-right: 36px; position: relative; }<br />
.nav-bar > li.has-flyout > a:first-child:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: #e6e6e6 transparent transparent transparent; position: absolute; right: 20px; top: 17px; }<br />
.nav-bar > li.has-flyout > a.flyout-toggle { border-left: 0 !important; position: absolute; right: 0; top: 0; padding: 22px; z-index: 2; display: block; }<br />
.nav-bar > li.has-flyout.is-touch > a:first-child { padding-right: 55px; }<br />
.nav-bar > li.has-flyout.is-touch > a.flyout-toggle { border-left: 1px dashed #666; }<br />
.nav-bar > li > a:first-child { position: relative; padding: 0 20px; display: block; text-decoration: none; font-size: 14px; }<br />
.nav-bar > li > input { margin: 0 10px; }<br />
.nav-bar.vertical { height: auto; margin-top: 0; }<br />
.nav-bar.vertical > li { float: none; border-bottom: none; }<br />
.nav-bar.vertical > li.has-flyout > a:first-child:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: transparent transparent transparent #e6e6e6; }<br />
.nav-bar.vertical > li .flyout { left: 100%; top: -1px; }<br />
.nav-bar.vertical > li .flyout.right { left: auto; right: 100%; }<br />
<br />
.flyout { background: #f2f2f2; padding: 20px; margin: 0; border: 1px solid #d9d9d9; position: absolute; top: 39px; left: -1px; width: 250px; z-index: 40; -webkit-box-shadow: 0 1px 5px rgba(0, 0, 0, 0.1); -moz-box-shadow: 0 1px 5px rgba(0, 0, 0, 0.1); box-shadow: 0 1px 5px rgba(0, 0, 0, 0.1); /* remove margin on any first-child element */ /* remove margin on last element */ }<br />
.flyout p { line-height: 1.2; font-size: 13px; }<br />
.flyout *:first-child { margin-top: 0; }<br />
.flyout *:last-child { margin-bottom: 0; }<br />
.flyout.small { width: 166.66667px; }<br />
.flyout.large { width: 437.5px; }<br />
.flyout.right { left: auto; right: -2px; }<br />
.flyout.up { top: auto; bottom: 39px; }<br />
<br />
ul.flyout, .nav-bar li ul { padding: 0; list-style: none; }<br />
ul.flyout li, .nav-bar li ul li { border-left: solid 3px #CCC; }<br />
ul.flyout li a, .nav-bar li ul li a { background: #f2f2f2; border: 1px solid #e6e6e6; border-width: 1px 1px 0 0; color: #555; display: block; font-size: 14px; height: auto; line-height: 1; padding: 15px 20px; -webkit-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
ul.flyout li a:hover, .nav-bar li ul li a:hover { background: #ebebeb; color: #333; }<br />
ul.flyout li.active, .nav-bar li ul li.active { margin-top: 0; border-top: 1px solid #4d4d4d; border-left: 4px solid #1a1a1a; }<br />
ul.flyout li.active a, .nav-bar li ul li.active a { background: #4d4d4d; border: none; color: #fff; height: auto; margin: 0; position: static; top: 0; -webkit-box-shadow: 0 0 0; -moz-box-shadow: 0 0 0; box-shadow: 0 0 0; }<br />
<br />
/* Mobile Styles */<br />
@media only screen and (max-device-width: 1280px) { .touch .nav-bar li.has-flyout > a { padding-right: 36px !important; } }<br />
@media only screen and (max-width: 1279px) and (min-width: 768px) { .touch .nav-bar li a { font-size: 13px; font-size: 1.3rem; }<br />
.touch .nav-bar li.has-flyout > a.flyout-toggle { padding: 20px !important; }<br />
.touch .nav-bar li.has-flyout > a { padding-right: 36px !important; } }<br />
@media only screen and (max-width: 767px) { .nav-bar { height: auto; }<br />
.nav-bar > li { float: none; display: block; border-right: none; }<br />
.nav-bar > li > a.main { text-align: left; border-top: 1px solid #ddd; border-right: none; }<br />
.nav-bar > li:first-child > a.main { border-top: none; }<br />
.nav-bar > li.has-flyout > a.flyout-toggle { position: absolute; right: 0; top: 0; padding: 22px; z-index: 2; display: block; }<br />
.nav-bar > li.has-flyout.is-touch > a.flyout-toggle span { content: ""; width: 0; height: 0; display: block; }<br />
.nav-bar > li.has-flyout > a.flyout-toggle:hover span { border-top-color: #141414; }<br />
.nav-bar.vertical > li.has-flyout > .flyout { left: 0; }<br />
.flyout { position: relative; width: 100% !important; top: auto; margin-right: -2px; border-width: 1px 1px 0 1px; }<br />
.flyout.right { float: none; right: auto; left: -1px; }<br />
.flyout.small, .flyout.large { width: 100% !important; }<br />
.flyout p:last-child { margin-bottom: 18px; } }<br />
/* CSS for jQuery Orbit Plugin 1.4.0 Maintained for Foundation. foundation.zurb.com Free to use under the MIT license. http://www.opensource.org/licenses/mit-license.php<br />
*/<br />
/* Container ---------------------- */<br />
div.orbit-wrapper { width: 1px; height: 1px; position: relative; }<br />
<br />
div.orbit { width: 1px; height: 1px; position: relative; overflow: hidden; margin-bottom: 17px; }<br />
<br />
div.orbit.with-bullets { margin-bottom: 40px; }<br />
<br />
div.orbit .orbit-slide { max-width: 100%; position: absolute; top: 0; left: 0; }<br />
<br />
div.orbit a.orbit-slide { border: none; line-height: 0; display: none; }<br />
<br />
div.orbit div.orbit-slide { width: 100%; height: 100%; }<br />
<br />
/* Note: If your slider only uses content or anchors, you're going to want to put the width and height declarations on the ".orbit>div" and "div.orbit>a" tags in addition to just the .orbit-wrapper */<br />
/* Timer ---------------------- */<br />
div.orbit-wrapper div.timer { width: 40px; height: 40px; overflow: hidden; position: absolute; top: 10px; right: 10px; opacity: .6; cursor: pointer; z-index: 31; }<br />
<br />
div.orbit-wrapper span.rotator { display: block; width: 40px; height: 40px; position: absolute; top: 0; left: -20px; background: url('https://static.igem.org/mediawiki/2013/7/72/CornellRotator-black.png') no-repeat; z-index: 3; }<br />
<br />
div.orbit-wrapper span.mask { display: block; width: 20px; height: 40px; position: absolute; top: 0; right: 0; z-index: 2; overflow: hidden; }<br />
<br />
div.orbit-wrapper span.rotator.move { left: 0; }<br />
<br />
div.orbit-wrapper span.mask.move { width: 40px; left: 0; background: url('https://static.igem.org/mediawiki/2013/8/8a/CornellTimer-black.png') repeat 0 0; }<br />
<br />
div.orbit-wrapper span.pause { display: block; width: 40px; height: 40px; position: absolute; top: 0; left: 0; background: url('https://static.igem.org/mediawiki/2013/e/e8/CornellPause-black.png') no-repeat; z-index: 4; opacity: 0; }<br />
<br />
div.orbit-wrapper span.pause.active { background: url('https://static.igem.org/mediawiki/2013/e/e8/CornellPause-black.png') no-repeat 0 -40px; }<br />
<br />
div.orbit-wrapper div.timer:hover span.pause, div.orbit-wrapper span.pause.active { opacity: 1; }<br />
<br />
/* Captions ---------------------- */<br />
.orbit-caption { display: none; font-family: "HelveticaNeue", "Helvetica-Neue", Helvetica, Arial, sans-serif; }<br />
<br />
.orbit-wrapper .orbit-caption { background: #000; background: rgba(0, 0, 0, 0.6); z-index: 30; color: #fff; text-align: center; padding: 7px 0; font-size: 13px; font-size: 1.3rem; position: absolute; right: 0; bottom: 0; width: 100%; }<br />
<br />
/* Directional Nav ---------------------- */<br />
div.orbit-wrapper div.slider-nav { display: block; }<br />
<br />
div.orbit-wrapper div.slider-nav span { width: 39px; height: 50px; text-indent: -9999px; position: absolute; z-index: 30; top: 50%; margin-top: -25px; cursor: pointer; }<br />
<br />
div.orbit-wrapper div.slider-nav span.right { background: url('https://static.igem.org/mediawiki/2013/3/3e/CornellRight-arrow.png'); background-size: 100%; right: 0; }<br />
<br />
div.orbit-wrapper div.slider-nav span.left { background: url('https://static.igem.org/mediawiki/2013/e/e7/Left-arrow.png'); background-size: 100%; left: 0; }<br />
<br />
.lt-ie9 div.orbit-wrapper div.slider-nav span.right { background: url('https://static.igem.org/mediawiki/2013/9/96/Right-arrow-small.png'); }<br />
.lt-ie9 div.orbit-wrapper div.slider-nav span.left { background: url('https://static.igem.org/mediawiki/2013/d/de/Left-arrow-small.png'); }<br />
<br />
/* Bullet Nav ---------------------- */<br />
ul.orbit-bullets { position: absolute; z-index: 30; list-style: none; bottom: -40px; left: 50%; margin-left: -50px; padding: 0; }<br />
<br />
ul.orbit-bullets li { float: left; margin-left: 5px; cursor: pointer; color: #999; text-indent: -9999px; background: url('https://static.igem.org/mediawiki/2013/3/3b/CornellBullets.png') no-repeat 4px 0; width: 13px; height: 12px; overflow: hidden; }<br />
<br />
ul.orbit-bullets li.active { color: #222; background-position: -8px 0; }<br />
<br />
ul.orbit-bullets li.has-thumb { background: none; width: 100px; height: 75px; }<br />
<br />
ul.orbit-bullets li.active.has-thumb { background-position: 0 0; border-top: 2px solid #000; }<br />
<br />
/* Fluid Layout ---------------------- */<br />
div.orbit img.fluid-placeholder { visibility: hidden; position: static; display: block; width: 100%; }<br />
<br />
div.orbit, div.orbit-wrapper { width: 100% !important; }<br />
<br />
ul.orbit-bullets { position: absolute; z-index: 30; list-style: none; bottom: -50px; left: 50%; margin-left: -50px; padding: 0; }<br />
<br />
ul.orbit-bullets li { float: left; margin-left: 5px; cursor: pointer; color: #999; text-indent: -9999px; background: url('https://static.igem.org/mediawiki/2013/3/3b/CornellBullets.png') no-repeat 4px 0; width: 13px; height: 12px; overflow: hidden; }<br />
<br />
ul.orbit-bullets li.has-thumb { background: none; width: 100px; height: 75px; }<br />
<br />
ul.orbit-bullets li.active { color: #222; background-position: -8px 0; }<br />
<br />
ul.orbit-bullets li.active.has-thumb { background-position: 0 0; border-top: 2px solid #000; }<br />
<br />
/* Correct timer in IE */<br />
.lt-ie9 .timer { display: none !important; }<br />
<br />
.lt-ie9 div.caption { background: transparent; filter: progid:DXImageTransform.Microsoft.gradient(startColorstr=#99000000,endColorstr=#99000000); zoom: 1; }<br />
<br />
/* CSS for jQuery Reveal Plugin Maintained for Foundation. foundation.zurb.com Free to use under the MIT license. http://www.opensource.org/licenses/mit-license.php */<br />
/* Reveal Modals ---------------------- */<br />
.reveal-modal-bg { position: fixed; height: 100%; width: 100%; background: #000; background: rgba(0, 0, 0, 0.45); z-index: 40; display: none; top: 0; left: 0; }<br />
<br />
.reveal-modal { background: white; visibility: hidden; display: none; top: 100px; left: 50%; margin-left: -260px; width: 520px; position: absolute; z-index: 41; padding: 30px; -webkit-box-shadow: 0 0 10px rgba(0, 0, 0, 0.4); -moz-box-shadow: 0 0 10px rgba(0, 0, 0, 0.4); box-shadow: 0 0 10px rgba(0, 0, 0, 0.4); }<br />
.reveal-modal *:first-child { margin-top: 0; }<br />
.reveal-modal *:last-child { margin-bottom: 0; }<br />
.reveal-modal .close-reveal-modal { font-size: 22px; font-size: 2.2rem; line-height: .5; position: absolute; top: 8px; right: 11px; color: #aaa; text-shadow: 0 -1px 1px rgba(0, 0, 0, 0.6); font-weight: bold; cursor: pointer; }<br />
.reveal-modal.small { width: 30%; margin-left: -10%; }<br />
.reveal-modal.medium { width: 40%; margin-left: -20%; }<br />
.reveal-modal.large { width: 60%; margin-left: -30%; }<br />
.reveal-modal.xlarge { width: 70%; margin-left: -35%; }<br />
.reveal-modal.expand { width: 90%; margin-left: -45%; }<br />
.reveal-modal .row { min-width: 0; }<br />
<br />
/* Mobile */<br />
@media only screen and (max-width: 767px) { .reveal-modal-bg { position: absolute; }<br />
.reveal-modal, .reveal-modal.small, .reveal-modal.medium, .reveal-modal.large, .reveal-modal.xlarge { width: 80%; top: 15px; left: 50%; margin-left: -40%; padding: 20px; height: auto; } }<br />
/* NOTES Close button entity is &#215;<br />
Example markup <div id="myModal" class="reveal-modal"> <h2>Awesome. I have it.</h2> <p class="lead">Your couch. I it's mine.</p> <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. In ultrices aliquet placerat. Duis pulvinar orci et nisi euismod vitae tempus lorem consectetur. Duis at magna quis turpis mattis venenatis eget id diam. </p> <a class="close-reveal-modal">&#215;</a> </div> */<br />
/* Requires globals.css app.js */<br />
/* Tabs ---------------------- */<br />
dl.tabs { border-bottom: solid 1px #e6e6e6; display: block; height: 40px; padding: 0; margin-bottom: 20px; }<br />
dl.tabs.contained { margin-bottom: 0; }<br />
dl.tabs dt { color: #b3b3b3; cursor: default; display: block; float: left; font-size: 12px; height: 40px; line-height: 40px; padding: 0 9px 0 20px; width: auto; text-transform: uppercase; }<br />
dl.tabs dt:first-child { padding: 0 9px 0 0; }<br />
dl.tabs dd { display: block; float: left; padding: 0; margin: 0; }<br />
dl.tabs dd a { color: #6f6f6f; display: block; font-size: 14px; height: 40px; line-height: 40px; padding: 0px 23.8px; }<br />
dl.tabs dd a:focus { font-weight: bold; color: #2ba6cb; }<br />
dl.tabs dd.active { border-top: 3px solid #2ba6cb; margin-top: -3px; }<br />
dl.tabs dd.active a { cursor: default; color: #3c3c3c; background: #fff; border-left: 1px solid #e6e6e6; border-right: 1px solid #e6e6e6; font-weight: bold; }<br />
dl.tabs dd:first-child { margin-left: 0; }<br />
dl.tabs.vertical { height: auto; border-bottom: 1px solid #e6e6e6; }<br />
dl.tabs.vertical dt, dl.tabs.vertical dd { float: none; height: auto; }<br />
dl.tabs.vertical dd { border-left: 3px solid #cccccc; }<br />
dl.tabs.vertical dd a { background: #f2f2f2; border: none; border: 1px solid #e6e6e6; border-width: 1px 1px 0 0; color: #555; display: block; font-size: 14px; height: auto; line-height: 1; padding: 15px 20px; -webkit-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
dl.tabs.vertical dd.active { margin-top: 0; border-top: 1px solid #4d4d4d; border-left: 4px solid #1a1a1a; }<br />
dl.tabs.vertical dd.active a { background: #4d4d4d; border: none; color: #fff; height: auto; margin: 0; position: static; top: 0; -webkit-box-shadow: 0 0 0; -moz-box-shadow: 0 0 0; box-shadow: 0 0 0; }<br />
dl.tabs.vertical dd:first-child a.active { margin: 0; }<br />
dl.tabs.pill { border-bottom: none; margin-bottom: 10px; }<br />
dl.tabs.pill dd { margin-right: 10px; }<br />
dl.tabs.pill dd:last-child { margin-right: 0; }<br />
dl.tabs.pill dd a { -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; background: #e6e6e6; height: 26px; line-height: 26px; color: #666; }<br />
dl.tabs.pill dd.active { border: none; margin-top: 0; }<br />
dl.tabs.pill dd.active a { background-color: #2ba6cb; border: none; color: #fff; }<br />
dl.tabs.pill.contained { border-bottom: solid 1px #eee; margin-bottom: 0; }<br />
dl.tabs.two-up dt a, dl.tabs.two-up dd a, dl.tabs.three-up dt a, dl.tabs.three-up dd a, dl.tabs.four-up dt a, dl.tabs.four-up dd a, dl.tabs.five-up dt a, dl.tabs.five-up dd a { padding: 0 17px; text-align: center; overflow: hidden; }<br />
dl.tabs.two-up dt, dl.tabs.two-up dd { width: 50%; }<br />
dl.tabs.three-up dt, dl.tabs.three-up dd { width: 33.33%; }<br />
dl.tabs.four-up dt, dl.tabs.four-up dd { width: 25%; }<br />
dl.tabs.five-up dt, dl.tabs.five-up dd { width: 20%; }<br />
<br />
ul.tabs-content { display: block; margin: 0 0 20px; padding: 0; }<br />
ul.tabs-content > li { display: none; }<br />
ul.tabs-content > li.active { display: block; }<br />
ul.tabs-content.contained { padding: 0; }<br />
ul.tabs-content.contained > li { border: solid 0 #e6e6e6; border-width: 0 1px 1px 1px; padding: 20px; }<br />
ul.tabs-content.contained.vertical > li { border-width: 1px 1px 1px 1px; }<br />
<br />
.no-js ul.tabs-content > li { display: block; }<br />
<br />
@media only screen and (max-width: 767px) { dl.tabs.mobile { width: auto; margin: 20px -20px 40px; height: auto; }<br />
dl.tabs.mobile dt, dl.tabs.mobile dd { float: none; height: auto; }<br />
dl.tabs.mobile dd a { display: block; width: auto; height: auto; padding: 18px 20px; line-height: 1; border: solid 0 #ccc; border-width: 1px 0 0; margin: 0; color: #555; background: #eee; font-size: 15px; font-size: 1.5rem; }<br />
dl.tabs.mobile dd a.active { height: auto; margin: 0; border-width: 1px 0 0; }<br />
.tabs.mobile { border-bottom: solid 1px #ccc; height: auto; }<br />
.tabs.mobile dd a { padding: 18px 20px; border: none; border-left: none; border-right: none; border-top: 1px solid #ccc; background: #fff; }<br />
.tabs.mobile dd a.active { border: none; background: #2ba6cb; color: #fff; margin: 0; position: static; top: 0; height: auto; }<br />
.tabs.mobile dd:first-child a.active { margin: 0; }<br />
dl.contained.mobile { margin-bottom: 0; }<br />
dl.contained.tabs.mobile dd a { padding: 18px 20px; }<br />
dl.tabs.mobile + ul.contained { margin-left: -20px; margin-right: -20px; border-width: 0 0 1px 0; } }<br />
/* Requires: globals.css */<br />
/* Table of Contents<br />
<br />
:: Visibility<br />
:: Alerts<br />
:: Labels<br />
:: Tooltips<br />
:: Panels<br />
:: Accordion<br />
:: Side Nav<br />
:: Sub Nav<br />
:: Pagination<br />
:: Breadcrumbs<br />
:: Lists<br />
:: Link Lists<br />
:: Keystroke Chars<br />
:: Video<br />
:: Tables<br />
:: Microformats<br />
<br />
*/<br />
/* Visibility Classes ---------------------- */<br />
/* Standard (large) display targeting */<br />
.show-for-small, .show-for-medium, .show-for-medium-down, .hide-for-large, .hide-for-large-up, .show-for-xlarge { display: none !important; }<br />
<br />
.hide-for-xlarge, .show-for-large, .show-for-large-up, .hide-for-small, .hide-for-medium, .hide-for-medium-down { display: block !important; }<br />
<br />
/* Very large display targeting */<br />
@media only screen and (min-width: 1441px) { .hide-for-small, .hide-for-medium, .hide-for-medium-down, .hide-for-large, .show-for-large-up, .show-for-xlarge { display: block !important; }<br />
.show-for-small, .show-for-medium, .show-for-medium-down, .show-for-large, .hide-for-large-up, .hide-for-xlarge { display: none !important; } }<br />
/* Medium display targeting */<br />
@media only screen and (max-width: 1279px) and (min-width: 768px) { .hide-for-small, .show-for-medium, .show-for-medium-down, .hide-for-large, .hide-for-large-up, .hide-for-xlarge { display: block !important; }<br />
.show-for-small, .hide-for-medium, .hide-for-medium-down, .show-for-large, .show-for-large-up, .show-for-xlarge { display: none !important; } }<br />
/* Small display targeting */<br />
@media only screen and (max-width: 767px) { .show-for-small, .hide-for-medium, .show-for-medium-down, .hide-for-large, .hide-for-large-up, .hide-for-xlarge { display: block !important; }<br />
.hide-for-small, .show-for-medium, .hide-for-medium-down, .show-for-large, .show-for-large-up, .show-for-xlarge { display: none !important; } }<br />
/* Orientation targeting */<br />
.show-for-landscape, .hide-for-portrait { display: block !important; }<br />
<br />
.hide-for-landscape, .show-for-portrait { display: none !important; }<br />
<br />
@media screen and (orientation: landscape) { .show-for-landscape, .hide-for-portrait { display: block !important; }<br />
.hide-for-landscape, .show-for-portrait { display: none !important; } }<br />
@media screen and (orientation: portrait) { .show-for-portrait, .hide-for-landscape { display: block !important; }<br />
.hide-for-portrait, .show-for-landscape { display: none !important; } }<br />
/* Touch-enabled device targeting */<br />
.show-for-touch { display: none !important; }<br />
<br />
.hide-for-touch { display: block !important; }<br />
<br />
.touch .show-for-touch { display: block !important; }<br />
<br />
.touch .hide-for-touch { display: none !important; }<br />
<br />
/* Specific overrides for elements that require something other than display: block */<br />
table.show-for-xlarge, table.show-for-large, table.hide-for-small, table.hide-for-medium { display: table !important; }<br />
<br />
@media only screen and (max-width: 1279px) and (min-width: 768px) { .touch table.hide-for-xlarge, .touch table.hide-for-large, .touch table.hide-for-small, .touch table.show-for-medium { display: table !important; } }<br />
@media only screen and (max-width: 767px) { table.hide-for-xlarge, table.hide-for-large, table.hide-for-medium, table.show-for-small { display: table !important; } }<br />
/* Alerts ---------------------- */<br />
div.alert-box { display: block; padding: 6px 7px 7px; font-weight: bold; font-size: 14px; color: white; background-color: #2ba6cb; border: 1px solid rgba(0, 0, 0, 0.1); margin-bottom: 12px; -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; text-shadow: 0 -1px rgba(0, 0, 0, 0.3); position: relative; }<br />
div.alert-box.success { background-color: #5da423; color: #fff; text-shadow: 0 -1px rgba(0, 0, 0, 0.3); }<br />
div.alert-box.alert { background-color: #c60f13; color: #fff; text-shadow: 0 -1px rgba(0, 0, 0, 0.3); }<br />
div.alert-box.secondary { background-color: #e9e9e9; color: #505050; text-shadow: 0 1px rgba(255, 255, 255, 0.3); }<br />
div.alert-box a.close { color: #333; position: absolute; right: 4px; top: -1px; font-size: 17px; opacity: 0.2; padding: 4px; }<br />
div.alert-box a.close:hover, div.alert-box a.close:focus { opacity: 0.4; }<br />
<br />
/* Labels ---------------------- */<br />
.label { padding: 1px 4px 2px; font-size: 12px; font-weight: bold; text-align: center; text-decoration: none; line-height: 1; white-space: nowrap; display: inline; position: relative; bottom: 1px; color: #fff; background: #2ba6cb; }<br />
.label.radius { -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
.label.round { padding: 1px 7px 2px; -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; }<br />
.label.alert { background-color: #c60f13; }<br />
.label.success { background-color: #5da423; }<br />
.label.secondary { background-color: #e9e9e9; color: #505050; }<br />
<br />
/* Tooltips ---------------------- */<br />
.has-tip { border-bottom: dotted 1px #cccccc; cursor: help; font-weight: bold; color: #333333; }<br />
.has-tip:hover { border-bottom: dotted 1px #196177; color: #2ba6cb; }<br />
.has-tip.tip-left, .has-tip.tip-right { float: none !important; }<br />
<br />
.tooltip { display: none; background: black; background: rgba(0, 0, 0, 0.85); position: absolute; color: white; font-weight: bold; font-size: 12px; font-size: 1.2rem; padding: 5px; z-index: 999; -webkit-border-radius: 4px; -moz-border-radius: 4px; border-radius: 4px; line-height: normal; }<br />
.tooltip > .nub { display: block; width: 0; height: 0; border: solid 5px; border-color: transparent transparent black transparent; border-color: transparent transparent rgba(0, 0, 0, 0.85) transparent; position: absolute; top: -10px; left: 10px; }<br />
.tooltip.tip-override > .nub { border-color: transparent transparent black transparent !important; border-color: transparent transparent rgba(0, 0, 0, 0.85) transparent !important; top: -10px !important; }<br />
.tooltip.tip-top > .nub { border-color: black transparent transparent transparent; border-color: rgba(0, 0, 0, 0.85) transparent transparent transparent; top: auto; bottom: -10px; }<br />
.tooltip.tip-left, .tooltip.tip-right { float: none !important; }<br />
.tooltip.tip-left > .nub { border-color: transparent transparent transparent black; border-color: transparent transparent transparent rgba(0, 0, 0, 0.85); right: -10px; left: auto; }<br />
.tooltip.tip-right > .nub { border-color: transparent black transparent transparent; border-color: transparent rgba(0, 0, 0, 0.85) transparent transparent; right: auto; left: -10px; }<br />
.tooltip.noradius { -webkit-border-radius: 0; -moz-border-radius: 0; -ms-border-radius: 0; -o-border-radius: 0; border-radius: 0; }<br />
.tooltip.opened { color: #2ba6cb !important; border-bottom: dotted 1px #196177 !important; }<br />
<br />
.tap-to-close { display: block; font-size: 10px; font-size: 1rem; color: #888888; font-weight: normal; }<br />
<br />
@media only screen and (max-width: 767px) { .tooltip { font-size: 14px; font-size: 1.4rem; line-height: 1.4; padding: 7px 10px 9px 10px; }<br />
.tooltip > .nub, .tooltip.top > .nub, .tooltip.left > .nub, .tooltip.right > .nub { border-color: transparent transparent black transparent; border-color: transparent transparent rgba(0, 0, 0, 0.85) transparent; top: -12px; left: 10px; } }<br />
/* Panels ---------------------- */<br />
.panel { background: #f2f2f2; border: solid 1px #e6e6e6; margin: 0 0 22px 0; padding: 20px; }<br />
.panel > :first-child { margin-top: 0; }<br />
.panel > :last-child { margin-bottom: 0; }<br />
.panel.callout { background: #2ba6cb; color: #fff; border-color: #2284a1; -webkit-box-shadow: inset 0px 1px 0px rgba(255, 255, 255, 0.5); -moz-box-shadow: inset 0px 1px 0px rgba(255, 255, 255, 0.5); box-shadow: inset 0px 1px 0px rgba(255, 255, 255, 0.5); }<br />
.panel.callout a { color: #fff; }<br />
.panel.callout .button { background: white; border: none; color: #2ba6cb; text-shadow: none; }<br />
.panel.callout .button:hover { background: rgba(255, 255, 255, 0.8); }<br />
.panel.radius { -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
<br />
/* Accordion ---------------------- */<br />
ul.accordion { margin: 0 0 22px 0; border-bottom: 1px solid #e9e9e9; }<br />
ul.accordion > li { list-style: none; margin: 0; padding: 0; border-top: 1px solid #e9e9e9; }<br />
ul.accordion > li .title { cursor: pointer; background: #f6f6f6; padding: 15px; margin: 0; position: relative; border-left: 1px solid #e9e9e9; border-right: 1px solid #e9e9e9; -webkit-transition: 0.15s background linear; -moz-transition: 0.15s background linear; -o-transition: 0.15s background linear; transition: 0.15s background linear; }<br />
ul.accordion > li .title h1, ul.accordion > li .title h2, ul.accordion > li .title h3, ul.accordion > li .title h4, ul.accordion > li .title h5 { margin: 0; }<br />
ul.accordion > li .title:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: transparent #9d9d9d transparent transparent; position: absolute; right: 15px; top: 21px; }<br />
ul.accordion > li .content { display: none; padding: 15px; }<br />
ul.accordion > li.active { border-top: 3px solid #2ba6cb; }<br />
ul.accordion > li.active .title { background: white; padding-top: 13px; }<br />
ul.accordion > li.active .title:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: #9d9d9d transparent transparent transparent; }<br />
ul.accordion > li.active .content { background: white; display: block; border-left: 1px solid #e9e9e9; border-right: 1px solid #e9e9e9; }<br />
<br />
/* Side Nav ---------------------- */<br />
ul.side-nav { display: block; list-style: none; margin: 0; padding: 17px 0; }<br />
ul.side-nav li { display: block; list-style: none; margin: 0 0 7px 0; }<br />
ul.side-nav li a { display: block; }<br />
ul.side-nav li.active a { color: #4d4d4d; font-weight: bold; }<br />
ul.side-nav li.inactive a { color: #b31b1b; font-weight: normal; }<br />
ul.side-nav li.divider { border-top: 1px solid #e6e6e6; height: 0; padding: 0; }<br />
<br />
/* Sub Navs http://www.zurb.com/article/292/how-to-create-simple-and-effective-sub-na ---------------------- */<br />
dl.sub-nav { display: block; width: auto; overflow: hidden; margin: -4px 0 18px -9px; padding-top: 4px; }<br />
dl.sub-nav dt, dl.sub-nav dd { float: left; display: inline; margin-left: 9px; margin-bottom: 10px; }<br />
dl.sub-nav dt { color: #999; font-weight: normal; }<br />
dl.sub-nav dd a { text-decoration: none; -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; }<br />
dl.sub-nav dd.active a { font-weight: bold; background: #2ba6cb; color: #fff; padding: 3px 9px; cursor: default; }<br />
<br />
/* Pagination ---------------------- */<br />
ul.pagination { display: block; height: 24px; margin-left: -5px; }<br />
ul.pagination li { float: left; display: block; height: 24px; color: #999; font-size: 14px; margin-left: 5px; }<br />
ul.pagination li a { display: block; padding: 1px 7px 1px; color: #555; }<br />
ul.pagination li:hover a, ul.pagination li a:focus { background: #e6e6e6; }<br />
ul.pagination li.unavailable a { cursor: default; color: #999; }<br />
ul.pagination li.unavailable:hover a, ul.pagination li.unavailable a:focus { background: transparent; }<br />
ul.pagination li.current a { background: #2ba6cb; color: white; font-weight: bold; cursor: default; }<br />
ul.pagination li.current a:hover { background: #2ba6cb; }<br />
<br />
/* Breadcrums ---------------------- */<br />
ul.breadcrumbs { display: block; background: #f6f6f6; padding: 6px 10px 7px; border: 1px solid #e9e9e9; -webkit-border-radius: 2px; -moz-border-radius: 2px; -ms-border-radius: 2px; -o-border-radius: 2px; border-radius: 2px; overflow: hidden; }<br />
ul.breadcrumbs li { margin: 0; padding: 0 12px 0 0; float: left; list-style: none; }<br />
ul.breadcrumbs li a, ul.breadcrumbs li span { text-transform: uppercase; font-size: 11px; font-size: 1.1rem; padding-left: 12px; }<br />
ul.breadcrumbs li:first-child a, ul.breadcrumbs li:first-child span { padding-left: 0; }<br />
ul.breadcrumbs li:before { content: "/"; color: #aaa; }<br />
ul.breadcrumbs li:first-child:before { content: " "; }<br />
ul.breadcrumbs li.current a { cursor: default; color: #333; }<br />
ul.breadcrumbs li:hover a, ul.breadcrumbs li a:focus { text-decoration: underline; }<br />
ul.breadcrumbs li.current:hover a, ul.breadcrumbs li.current a:focus { text-decoration: none; }<br />
ul.breadcrumbs li.unavailable a { color: #999; }<br />
ul.breadcrumbs li.unavailable:hover a, ul.breadcrumbs li.unavailable a:focus { text-decoration: none; color: #999; cursor: default; }<br />
<br />
/* Link List */<br />
ul.link-list { margin: 0 0 17px -22px; padding: 0; list-style: none; overflow: hidden; }<br />
ul.link-list li { list-style: none; float: left; margin-left: 22px; display: block; }<br />
ul.link-list li a { display: block; }<br />
<br />
/* Keytroke Characters ---------------------- */<br />
.keystroke, kbd { font-family: "Consolas", "Menlo", "Courier", monospace; font-size: 13px; padding: 2px 4px 0px; margin: 0; background: #ededed; border: solid 1px #dbdbdb; -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
<br />
/* Video - Mad props to http://www.alistapart.com/articles/creating-intrinsic-ratios-for-video/ ---------------------- */<br />
.flex-video { position: relative; padding-top: 25px; padding-bottom: 67.5%; height: 0; margin-bottom: 16px; overflow: hidden; }<br />
.flex-video.widescreen { padding-bottom: 57.25%; }<br />
.flex-video.vimeo { padding-top: 0; }<br />
.flex-video iframe, .flex-video object, .flex-video embed, .flex-video video { position: absolute; top: 0; left: 0; width: 100%; height: 100%; }<br />
<br />
@media only screen and (max-device-width: 800px), only screen and (device-width: 1024px) and (device-height: 600px), only screen and (width: 1280px) and (orientation: landscape), only screen and (device-width: 800px), only screen and (max-width: 767px) { .flex-video { padding-top: 0; } }<br />
/* Tables ---------------------- */<br />
table { background: #fff; -moz-border-radius: 3px; -webkit-border-radius: 3px; border-radius: 3px; margin: 0 0 18px; border: 1px solid; }<br />
<br />
table thead, table tfoot { background: #f5f5f5; }<br />
<br />
table thead tr th, table tfoot tr th, table tbody tr td, table tr td, table tfoot tr td { font-size: 14px; line-height: 18px; text-align: left; }<br />
<br />
table thead tr th, table tfoot tr td { padding: 8px 10px 9px; font-size: 16px; font-weight: bold; color: #222; }<br />
<br />
table thead tr th:first-child, table tfoot tr td:first-child { border-left: none; }<br />
<br />
table thead tr th:last-child, table tfoot tr td:last-child { border-right: none; }<br />
<br />
table tbody tr.even, table tbody tr.alt { background: #f9f9f9; }<br />
<br />
table tbody tr:nth-child(even) { background: #f2f2f2; }<br />
<br />
table tbody tr td { color: #333; padding: 9px 10px; vertical-align: top; border: 1px solid; }<br />
<br />
/* Microformats ---------------------- */<br />
ul.vcard { display: inline-block; margin: 0 0 12px 0; border: 1px solid #ddd; padding: 10px; }<br />
ul.vcard li { margin: 0; display: block; }<br />
ul.vcard li.fn { font-weight: bold; font-size: 15px; font-size: 1.5rem; }<br />
<br />
p.vevent span.summary { font-weight: bold; }<br />
p.vevent abbr { cursor: default; text-decoration: none; font-weight: bold; border: none; padding: 0 1px; }</div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/notebook/plasmidsTeam:Cornell/notebook/plasmids2013-09-28T03:04:22Z<p>R.Lizarralde: </p>
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<table class="tableizer-table" rules="all"><br />
<tr class="tableizer-firstrow"><th>Plasmid Name</th><th>Part</th><th>Contents/Description</th><th>Origin</th><th>Backbone</th><th>Antibiotic Resistance</th><th>Antifungal Resistance</th></tr><br />
<tr><td>pC13A</td><td>BBa_K523022</td><td>crtEIB</td><td>Kit Plate 1 8P</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13B</td><td>BBa_K539119</td><td>crtY</td><td>Kit Plate 1 12K</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pA13C</td><td>pIVEX2.3d</td><td>T7 promoter/terminator</td><td>Luo Lab</td><td>pIVEX2.3d</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pAK13D</td><td>BBa_I712074</td><td>T7 promoter</td><td>Kit Plate 5 6N</td><td>pSB1AK8</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13E</td><td>BBa_K145001</td><td>T7 polymerase</td><td>Kit Plate 4 2C</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13F</td><td>BBa_J04450</td><td>mRFP in pSB1C3</td><td>T.E. Kit</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAh13G</td><td>pUCATPH</td><td>PtrpC, hph, TtrpC</td><td>Turgeon lab</td><td>pUCATPH</td><td>Ampicillin</td><td>Hygromycin</td></tr><br />
<tr><td>pAg13H</td><td>pNG</td><td>Pgpd (C. h.), nptII</td><td>Turgeon lab</td><td>pNG</td><td>Ampicillin</td><td>Geneticin</td></tr><br />
<tr><td>pC13I</td><td>BBa_K1021000</td><td>crtE</td><td>A</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13J</td><td>invalid</td><td>rbs + crtE</td><td>A</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13K</td><td>BBa_K118003</td><td>crtI</td><td>Kit Plate 3 2B</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13L</td><td>BBa_K118005</td><td>rbs + crtI</td><td>Kit Plate 4 4A</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13M</td><td>BBa_K118002</td><td>crtB</td><td>Kit Plate 3 17N</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13N</td><td>BBa_K118006</td><td>rbs + crtB</td><td>Kit Plate 3 17P</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13O</td><td>BBa_I742155</td><td>rbs + crtY</td><td>Kit Plate 5 17P</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13P</td><td>BBa_K1021007</td><td>PtrpC</td><td>G</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13Q</td><td>BBa_K1021008</td><td>TtrpC</td><td>G</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pCg13R</td><td>invalid</td><td>nptII</td><td>H</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pCg13S</td><td>BBa_K1021001</td><td>nptIIBB</td><td>R</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pAb13T</td><td>pBARGPE1</td><td>PtrpC, bar, TtrpC, PgpdA, lox</td><td>Fungal Genetics Stock Center</td><td>pBARGPE1</td><td>Ampicillin</td><td>Phosphonothricin</td></tr><br />
<tr><td>pCg13U</td><td>invalid</td><td>nptIIBB + TtrpC</td><td>S, Q</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pCh13V</td><td>invalid</td><td>hph</td><td>G, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pCh13W</td><td>BBa_K1021002</td><td>hphBB</td><td>V</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pCp13X</td><td>BBa_K1021003</td><td>bar</td><td>T, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Phosphonothricin</td></tr><br />
<tr><td>pCg13Y</td><td>BBa_K1021015</td><td>PtrpC + nptIIBB</td><td>P, S</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13Z</td><td>BBa_K082003</td><td>GFP(+LVA)</td><td>Kit Plate 3 20N</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13AA</td><td>invalid</td><td>PT7 + crtE</td><td>D, A</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AB</td><td>invalid</td><td>PT7 + crtB</td><td>D, M</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AC</td><td>invalid</td><td>PT7 + crtI</td><td>D, K</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AD</td><td>invalid</td><td>PT7 + crtY</td><td>D, B</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13AE</td><td>BBa_K1021016</td><td>PT7 + crtE</td><td>F, AA</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AF</td><td>BBa_K1021017</td><td>PT7 + crtB</td><td>F, AB</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AG</td><td>BBa_K1021018</td><td>PT7 + crtI</td><td>F, AC</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AH</td><td>BBa_K1021019</td><td>PT7 + crtY</td><td>F, AD</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AI</td><td>BBa_J61046</td><td>lox site</td><td>Kit Plate 3 3L</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pA13AJ</td><td>BBa_J61046</td><td>lox site</td><td>Kit Plate 5 24B</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13AK</td><td>BBa_E1010</td><td>mRFP cds only</td><td>Kit Plate 3 12N</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pK13AL</td><td>BBa_E1010</td><td>mRFP cds only</td><td>Kit Plate 5 18F</td><td>pSB2K3</td><td>Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pA13AM</td><td>BBa_J23100</td><td>strongest Anderson constitutive promoter with mRFP downstream</td><td>Kit Plate 5 18C</td><td>BBa_J61002</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AN</td><td>invalid</td><td>PT7+GFP</td><td>D, Z</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AO</td><td>invalid</td><td>PT7+mRFP</td><td>D, AK/AL</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AP</td><td>invalid</td><td>PT7+bar</td><td>D, X</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>Phosphonothricin</td></tr><br />
<tr><td>pC13AQ</td><td>BBa_K1021020</td><td>PT7+GFP</td><td>AN, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AR</td><td>BBa_K1021021</td><td>PT7+mRFP</td><td>AO, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AS</td><td>BBa_K1021022</td><td>PT7+bar</td><td>AP, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Phosphonothricin</td></tr><br />
<tr><td>pC13AT</td><td>BBa_K1021023</td><td>PtrpC+GFP</td><td>P, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AU</td><td>BBa_K1021024</td><td>PtrpC+mRFP</td><td>P, AK/AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AV</td><td>BBa_K1021025</td><td>PtrpC+bar</td><td>P, X</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Phosphonothricin</td></tr><br />
<tr><td>pA13AW</td><td>invalid</td><td>Anderson promoter + T7Pol</td><td>AM, E</td><td>BBa_J61002</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pK13AX</td><td>invalid</td><td>Anderson promoter + T7Pol</td><td>AW, AL (backbone)</td><td>pSB2K3</td><td>Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13AY</td><td>unsubmitted</td><td>PtrpC+T7Pol</td><td>P, E</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AZ</td><td>BBa_K1021026</td><td>Anderson promoter + T7Pol</td><td>AW, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BA</td><td>BBa_K1021004</td><td>afp1</td><td>G-block</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13BB</td><td>invalid</td><td>PT7+nptIIBB</td><td>D, S</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>Geneticin</td></tr><br />
<tr><td>pC13BC</td><td>BBa_K1021032</td><td>PT7+nptIIBB</td><td>BB, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13BD</td><td>unsubmitted</td><td>PtrpC+crtI</td><td>P, K</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pA13BE</td><td>pHNU3pelA</td><td>PpelA, flanked by KpnI/BamHI</td><td>Turgeon lab</td><td>pHPG</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13BF</td><td>BBa_K1021009</td><td>PpelA</td><td>BE, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BG</td><td>BBa_K1021010</td><td>A. nidulans PgpdA</td><td>T, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BH</td><td>invalid</td><td>G. lucidum PgpdA</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BI</td><td>unsubmitted</td><td>G. lucidum PgpdA (BB)</td><td>BH</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BJ</td><td>BBa_K1021011</td><td>100bp left region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BK</td><td>BBa_K1021012</td><td>200bp left region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BL</td><td>BBa_K1021013</td><td>100bp right region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BM</td><td>BBa_K1021014</td><td>200bp right region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BN</td><td>unsubmitted</td><td>lox [cutsite 1] [cutsite 2] lox</td><td>AI</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BO</td><td>abandoned</td><td>short homology regions flanking two unique cutsites</td><td>BJ, BL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BP</td><td>abandoned</td><td>long homology regions flanking two unique cutsites</td><td>BK, BM</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BQ</td><td>BBa_K1021027</td><td>A. nidulans PgpdA + nptIIBB</td><td>BG, S</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13BR</td><td>BBa_K1021028</td><td>A. nidulans PgpdA + hphBB</td><td>BG, W</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13BS</td><td>unsubmitted</td><td>G. lucidum PgpdABB + nptIIBB</td><td>BI, S</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13BT</td><td>unsubmitted</td><td>G. lucidum PgpdABB + hphBB</td><td>BI, W</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pAK13BU</td><td>invalid</td><td>PT7 + hphBB</td><td>D, W</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>Hygromycin</td></tr><br />
<tr><td>pC13BV</td><td>BBa_K1021033</td><td>PT7 + hphBB</td><td>BU, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pA13BW</td><td>pBS185CMV-Cre</td><td>pBS185 CMV Cre</td><td>Addgene</td><td>pBS185</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13BX</td><td>BBa_K1021005</td><td>Cre recombinase</td><td>BW</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BY</td><td>unsubmitted</td><td>PpelA + Cre</td><td>BF, BX</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BZ</td><td>unsubmitted</td><td>PpelA + GFP</td><td>BF, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CA</td><td>unsubmitted</td><td>PpelA + mRFP</td><td>BF, AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CB</td><td>unsubmitted</td><td>PT7 + GFP + PtrpC + nptIIBB</td><td>Y, AQ</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13CC</td><td>BBa_K1021029</td><td>PtrpC + nptIIBB + TtrpC</td><td>Y, Q</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13CD</td><td>BBa_K1021030</td><td>PtrpC + hphBB</td><td>P, W</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13CE</td><td>BBa_K1021034</td><td>PtrpC + hphBB +TtrpC</td><td>CD, Q</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13CF</td><td>unsubmitted</td><td>A. n. PgpdA + GFP</td><td>BG, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CG</td><td>BBa_K1021031</td><td>A. n. PgpdA + mRFP</td><td>BG, AK/AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CH</td><td>unsubmitted</td><td>G. l. PgpdA + GFP</td><td>BH, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CI</td><td>unsubmitted</td><td>G. I. PgpdA + mRFP</td><td>BH, AK/AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CJ</td><td>BBa_I742111</td><td>Limonene synthase</td><td>Kit plate 4</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13CK</td><td>unsubmitted</td><td>PT7+Limonene Synthase</td><td>CJ, D</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13CL</td><td>BBa_K1021006</td><td>Cht1_2 in pSB1C3</td><td>IDT Geneblock</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CM</td><td>unsubmitted</td><td>PpelA + Cht1_2</td><td>CL, BF</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pS13CN</td><td>BBa_K112306</td><td>Lambda Holin</td><td>Kit plate 3 2012 BBa_K112306</td><td>BBa_K112950</td><td>Spectinomycin</td><td>N/A</td></tr><br />
<tr><td>pC13CO</td><td>unsubmitted</td><td>Lambda Holin in pSB1C3</td><td>CN</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CP</td><td>unsubmitted</td><td>PpelA+Lambda Holin</td><td>CO</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13CQ</td><td>invalid</td><td>PT7 + rbs + crtE</td><td>D, J</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13CR</td><td>invalid</td><td>PT7 + rbs + crtB</td><td>D, N</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13CS</td><td>invalid</td><td>PT7 + rbs + crtI</td><td>D, L</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13CT</td><td>invalid</td><td>PT7 + rbs + crtY</td><td>D, O</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13CU</td><td>unsubmitted</td><td>PT7 + rbs + crtE</td><td>F, CQ</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CV</td><td>unsubmitted</td><td>PT7 + rbs + crtB</td><td>F, CR</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CW</td><td>unsubmitted</td><td>PT7 + rbs + crtI</td><td>F, CS</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CX</td><td>unsubmitted</td><td>PT7 + rbs + crtY</td><td>F, CT</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13CY</td><td>invalid</td><td>PT7 + rbs + GFP</td><td>D, DB</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pA13CZ</td><td>BBa_B0034</td><td>RBS (strong)</td><td>Kit Plate 5, Well 2M</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pAK13DA</td><td>invalid</td><td>PT7 + rbs + mRFP</td><td>D, DC</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pA13DB</td><td>invalid</td><td>RBS+GFP</td><td>CZ, Z</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pA13DC</td><td>invalid</td><td>RBS+mRFP</td><td>CZ, AK</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13DD</td><td>unsubmitted</td><td>PT7+Limonene Synthase</td><td>F, CK</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DE</td><td>unsubmitted</td><td>lox-PtrpC-nptIIBB-lox</td><td>Y, BN</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13DF</td><td>unsubmitted</td><td>lox-A.PgpdA-nptIIBB-lox</td><td>BN, BQ</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13DG</td><td>unsubmitted</td><td>lox-A.PgpdA-hphBB-lox</td><td>BN, BR</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13DH</td><td>unsubmitted</td><td>lox-PtrpC-hphBB-lox</td><td>BN, CD</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13DI</td><td>unsubmitted</td><td>PtrpC + hphBB + GFP</td><td>CD, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13DJ</td><td>unsubmitted</td><td>PT7 + crtB + PT7 + crtI</td><td>AF, AG</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DK</td><td>unsubmitted</td><td>PT7 + crtE + PT7 + crtB + PT7 + crtI</td><td>DJ, AE</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DL</td><td>unsubmitted</td><td>PT7 + crtE + PT7 + crtB + PT7 + crtI + PT7 + nptIIBB</td><td>DK, BC</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13DM</td><td>unsubmitted</td><td>PT7 + rbs + crtB + PT7 + rbs + crtI</td><td>CV, CW</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DN</td><td>unsubmitted</td><td>PT7 + rbs + crtE + PT7 + rbs + crtB + PT7 + rbs + crtI</td><td>DM, CU</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DO</td><td>unsubmitted</td><td>Gan. PgpdA + crtE</td><td>BI, I</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DP</td><td>unsubmitted</td><td>Asp. PgpdA + crtB</td><td>BG, M</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DQ</td><td>unsubmitted</td><td>Asp. PgpdA + crtB + PtrpC + crtI</td><td>DP, BD</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DR</td><td>unsubmitted</td><td>Gan. PgpdA + crtE + Asp. PgpdA + crtB + PtrpC + crtI</td><td>DQ, DO</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DS</td><td>unsubmitted</td><td>Gan. PgpdA + crtE + Asp. PgpdA + crtB + PtrpC + crtI + PtrpC + nptII</td><td>DS, DR</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
</table><br />
</div><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/stylesheets/foundationTeam:Cornell/stylesheets/foundation2013-09-28T03:04:05Z<p>R.Lizarralde: </p>
<hr />
<div>.c-1, .c-2, .c-3, .c-4, .c-5, .c-6, .c-7, .c-8, .c-9, .c-10, .c-11, .c-12 { float: left; }<br />
<br />
.c-1, .c-2, .c-3, .c-4, .c-5, .c-6, .c-7, .c-8, .c-9, .c-10, .c-11, .c-12 { position: relative; min-height: 1px; padding: 0 15px; }<br />
<br />
.c-1 { width: 8.33333%; }<br />
<br />
.c-2 { width: 16.66667%; }<br />
<br />
.c-3 { width: 25%; }<br />
<br />
.c-4 { width: 33.33333%; }<br />
<br />
.c-5 { width: 41.66667%; }<br />
<br />
.c-6 { width: 50%; }<br />
<br />
.c-7 { width: 58.33333%; }<br />
<br />
.c-8 { width: 66.66667%; }<br />
<br />
.c-9 { width: 75%; }<br />
<br />
.c-10 { width: 83.33333%; }<br />
<br />
.c-11 { width: 91.66667%; }<br />
<br />
.c-12 { width: 100%; }<br />
<br />
/* Requires: normalize.css */<br />
/* Global Reset & Standards ---------------------- */<br />
* { -webkit-box-sizing: border-box; -moz-box-sizing: border-box; box-sizing: border-box; }<br />
<br />
html { font-size: 62.5%; }<br />
<br />
body { background: white; font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-size: 14px; line-height: 1; color: #222222; position: relative; -webkit-font-smoothing: antialiased; }<br />
<br />
/* Links ---------------------- */<br />
a { color: #2ba6cb; text-decoration: none; line-height: inherit; }<br />
<br />
a:hover { color: #2795b6; }<br />
<br />
a:focus { color: #2ba6cb; outline: none; }<br />
<br />
p a, p a:visited { line-height: inherit; }<br />
<br />
/* Misc ---------------------- */<br />
.left { float: left; }<br />
<br />
.right { float: right; }<br />
<br />
.text-left { text-align: left; }<br />
<br />
.text-right { text-align: right; }<br />
<br />
.text-center { text-align: center; }<br />
<br />
.hide { display: none; }<br />
<br />
.highlight { background: #ffff99; }<br />
<br />
#googlemap img, object, embed { max-width: none; }<br />
<br />
#map_canvas embed { max-width: none; }<br />
<br />
#map_canvas img { max-width: none; }<br />
<br />
#map_canvas object { max-width: none; }<br />
<br />
/* Reset for strange margins by default on <figure> elements */<br />
figure { margin: 0; }<br />
<br />
/* Base Type Styles Using Modular Scale ---------------------- */<br />
body, div, dl, dt, dd, ul, ol, li, h1, h2, h3, h4, h5, h6, pre, form, fieldset, p, blockquote, th, td { margin: 0; padding: 0; font-size: 14px; }<br />
<br />
p { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-weight: normal; font-size: 14px; line-height: 1.6; margin-bottom: 17px; }<br />
p.lead { font-size: 17.5px; line-height: 1.6; margin-bottom: 17px; }<br />
<br />
aside p { font-size: 13px; line-height: 1.35; font-style: italic; }<br />
<br />
h1, h2, h3, h4, h5, h6 { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; text-rendering: optimizeLegibility; line-height: 1.1; margin-bottom: 14px; margin-top: 14px; }<br />
h1 small, h2 small, h3 small, h4 small, h5 small, h6 small { font-size: 60%; color: #6f6f6f; line-height: 0; }<br />
<br />
h1 { font-size: 44px; }<br />
<br />
h2 { font-size: 37px; }<br />
<br />
h3 { font-size: 27px; }<br />
<br />
h4 { font-size: 23px; }<br />
<br />
h5 { font-size: 17px; }<br />
<br />
h6 { font-size: 14px; }<br />
<br />
hr { border: solid #ddd; border-width: 1px 0 0; clear: both; margin: 22px 0 21px; height: 0; }<br />
<br />
.subheader { line-height: 1.3; color: #6f6f6f; font-weight: 300; margin-bottom: 17px; }<br />
<br />
em, i { font-style: italic; line-height: inherit; }<br />
<br />
strong, b { font-weight: bold; line-height: inherit; }<br />
<br />
small { font-size: 60%; line-height: inherit; }<br />
<br />
code { font-weight: bold; background: #ffff99; }<br />
<br />
/* Lists ---------------------- */<br />
ul, ol { font-size: 14px; line-height: 1.6; margin-bottom: 17px; list-style-position: inside; }<br />
<br />
ul li ul, ul li ol { margin-left: 20px; margin-bottom: 0; }<br />
ul.square, ul.circle, ul.disc { margin-left: 17px; }<br />
ul.square { list-style-type: square; }<br />
ul.square li ul { list-style: inherit; }<br />
ul.circle { list-style-type: circle; }<br />
ul.circle li ul { list-style: inherit; }<br />
ul.disc { list-style-type: disc; }<br />
ul.disc li ul { list-style: inherit; }<br />
ul.no-bullet { list-style: none; }<br />
ul.large li { line-height: 21px; }<br />
<br />
ol li ul, ol li ol { margin-left: 20px; margin-bottom: 0; }<br />
<br />
/* Blockquotes ---------------------- */<br />
blockquote, blockquote p { line-height: 1.5; color: #6f6f6f; }<br />
<br />
blockquote { margin: 0 0 17px; padding: 9px 20px 0 19px; border-left: 1px solid #ddd; }<br />
blockquote cite { display: block; font-size: 13px; color: #555555; }<br />
blockquote cite:before { content: "\2014 \0020"; }<br />
blockquote cite a, blockquote cite a:visited { color: #555555; }<br />
<br />
abbr, acronym { text-transform: uppercase; font-size: 90%; color: #222222; border-bottom: 1px solid #ddd; cursor: help; }<br />
<br />
abbr { text-transform: none; }<br />
<br />
/* Print styles. Inlined to avoid required HTTP connection: www.phpied.com/delay-loading-your-print-css/ Credit to Paul Irish and HTML5 Boilerplate (html5boilerplate.com)<br />
*/<br />
.print-only { display: none !important; }<br />
<br />
@media print { * { background: transparent !important; color: black !important; box-shadow: none !important; text-shadow: none !important; filter: none !important; -ms-filter: none !important; }<br />
/* Black prints faster: h5bp.com/s */<br />
a, a:visited { text-decoration: underline; }<br />
a[href]:after { content: " (" attr(href) ")"; }<br />
abbr[title]:after { content: " (" attr(title) ")"; }<br />
.ir a:after, a[href^="javascript:"]:after, a[href^="#"]:after { content: ""; }<br />
/* Don't show links for images, or javascript/internal links */<br />
pre, blockquote { border: 1px solid #999; page-break-inside: avoid; }<br />
thead { display: table-header-group; }<br />
/* h5bp.com/t */<br />
tr, img { page-break-inside: avoid; }<br />
img { max-width: 100% !important; }<br />
@page { margin: 0.5cm; }<br />
p, h2, h3 { orphans: 3; widows: 3; }<br />
h2, h3 { page-break-after: avoid; }<br />
.hide-on-print { display: none !important; }<br />
.print-only { display: block !important; } }<br />
/* Requires globals.css */<br />
/* Standard Forms ---------------------- */<br />
form { margin: 0 0 19.41641px; }<br />
<br />
.row form .row { margin: 0 -6px; }<br />
.row form .row .column, .row form .row .columns { padding: 0 6px; }<br />
.row form .row.collapse { margin: 0; }<br />
.row form .row.collapse .column, .row form .row.collapse .columns { padding: 0; }<br />
<br />
label { font-size: 14px; color: #4d4d4d; cursor: pointer; display: block; font-weight: 500; margin-bottom: 3px; }<br />
label.right { float: none; text-align: right; }<br />
label.inline { line-height: 32px; margin: 0 0 12px 0; }<br />
<br />
@media only screen and (max-width: 767px) { label.right { text-align: left; } }<br />
.prefix, .postfix { display: block; position: relative; z-index: 2; text-align: center; width: 100%; padding-top: 0; padding-bottom: 0; height: 32px; line-height: 31px; }<br />
<br />
a.button.prefix, a.button.postfix { padding-left: 0; padding-right: 0; text-align: center; }<br />
<br />
span.prefix, span.postfix { background: #f2f2f2; border: 1px solid #cccccc; }<br />
<br />
.prefix { left: 2px; -moz-border-radius-topleft: 2px; -webkit-border-top-left-radius: 2px; border-top-left-radius: 2px; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; }<br />
<br />
.postfix { right: 2px; -moz-border-radius-topright: 2px; -webkit-border-top-right-radius: 2px; border-top-right-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
input[type="text"], input[type="password"], input[type="date"], input[type="datetime"], input[type="email"], input[type="number"], input[type="search"], input[type="tel"], input[type="time"], input[type="url"], textarea { border: 1px solid #cccccc; -webkit-border-radius: 2px; -moz-border-radius: 2px; -ms-border-radius: 2px; -o-border-radius: 2px; border-radius: 2px; -webkit-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); -moz-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); color: rgba(0, 0, 0, 0.75); display: block; font-size: 14px; margin: 0 0 12px 0; padding: 6px; height: 32px; width: 100%; -webkit-transition: all 0.15s linear; -moz-transition: all 0.15s linear; -o-transition: all 0.15s linear; transition: all 0.15s linear; }<br />
input[type="text"].oversize, input[type="password"].oversize, input[type="date"].oversize, input[type="datetime"].oversize, input[type="email"].oversize, input[type="number"].oversize, input[type="search"].oversize, input[type="tel"].oversize, input[type="time"].oversize, input[type="url"].oversize, textarea.oversize { font-size: 17px; padding: 4px 6px; }<br />
input[type="text"]:focus, input[type="password"]:focus, input[type="date"]:focus, input[type="datetime"]:focus, input[type="email"]:focus, input[type="number"]:focus, input[type="search"]:focus, input[type="tel"]:focus, input[type="time"]:focus, input[type="url"]:focus, textarea:focus { background: #fafafa; outline: none !important; border-color: #b3b3b3; }<br />
input[type="text"][disabled], input[type="password"][disabled], input[type="date"][disabled], input[type="datetime"][disabled], input[type="email"][disabled], input[type="number"][disabled], input[type="search"][disabled], input[type="tel"][disabled], input[type="time"][disabled], input[type="url"][disabled], textarea[disabled] { background-color: #ddd; }<br />
<br />
textarea { height: auto; }<br />
<br />
select { width: 100%; }<br />
<br />
/* Fieldsets */<br />
fieldset { border: solid 1px #ddd; border-radius: 3px; -webkit-border-radius: 3px; -moz-border-radius: 3px; padding: 12px 12px 0; margin: 18px 0; }<br />
fieldset legend { font-weight: bold; background: white; padding: 0 3px; margin: 0 0 0 -3px; }<br />
<br />
/* Errors */<br />
.error input, input.error, .error textarea, textarea.error { border-color: #c60f13; background-color: rgba(198, 15, 19, 0.1); }<br />
<br />
.error label, label.error { color: #c60f13; }<br />
<br />
.error small, small.error { display: block; padding: 6px 4px; margin-top: -13px; margin-bottom: 12px; background: #c60f13; color: #fff; font-size: 12px; font-size: 1.2rem; font-weight: bold; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
@media only screen and (max-width: 767px) { input[type="text"].one, input[type="password"].one, input[type="date"].one, input[type="datetime"].one, input[type="email"].one, input[type="number"].one, input[type="search"].one, input[type="tel"].one, input[type="time"].one, input[type="url"].one, textarea.one, .row textarea.one { width: 100% !important; }<br />
input[type="text"].two, .row input[type="text"].two, input[type="password"].two, .row input[type="password"].two, input[type="date"].two, .row input[type="date"].two, input[type="datetime"].two, .row input[type="datetime"].two, input[type="email"].two, .row input[type="email"].two, input[type="number"].two, .row input[type="number"].two, input[type="search"].two, .row input[type="search"].two, input[type="tel"].two, .row input[type="tel"].two, input[type="time"].two, .row input[type="time"].two, input[type="url"].two, .row input[type="url"].two, textarea.two, .row textarea.two { width: 100% !important; }<br />
input[type="text"].three, .row input[type="text"].three, input[type="password"].three, .row input[type="password"].three, input[type="date"].three, .row input[type="date"].three, input[type="datetime"].three, .row input[type="datetime"].three, input[type="email"].three, .row input[type="email"].three, input[type="number"].three, .row input[type="number"].three, input[type="search"].three, .row input[type="search"].three, input[type="tel"].three, .row input[type="tel"].three, input[type="time"].three, .row input[type="time"].three, input[type="url"].three, .row input[type="url"].three, textarea.three, .row textarea.three { width: 100% !important; }<br />
input[type="text"].four, .row input[type="text"].four, input[type="password"].four, .row input[type="password"].four, input[type="date"].four, .row input[type="date"].four, input[type="datetime"].four, .row input[type="datetime"].four, input[type="email"].four, .row input[type="email"].four, input[type="number"].four, .row input[type="number"].four, input[type="search"].four, .row input[type="search"].four, input[type="tel"].four, .row input[type="tel"].four, input[type="time"].four, .row input[type="time"].four, input[type="url"].four, .row input[type="url"].four, textarea.four, .row textarea.four { width: 100% !important; }<br />
input[type="text"].five, .row input[type="text"].five, input[type="password"].five, .row input[type="password"].five, input[type="date"].five, .row input[type="date"].five, input[type="datetime"].five, .row input[type="datetime"].five, input[type="email"].five, .row input[type="email"].five, input[type="number"].five, .row input[type="number"].five, input[type="search"].five, .row input[type="search"].five, input[type="tel"].five, .row input[type="tel"].five, input[type="time"].five, .row input[type="time"].five, input[type="url"].five, .row input[type="url"].five, textarea.five, .row textarea.five { width: 100% !important; }<br />
input[type="text"].six, .row input[type="text"].six, input[type="password"].six, .row input[type="password"].six, input[type="date"].six, .row input[type="date"].six, input[type="datetime"].six, .row input[type="datetime"].six, input[type="email"].six, .row input[type="email"].six, input[type="number"].six, .row input[type="number"].six, input[type="search"].six, .row input[type="search"].six, input[type="tel"].six, .row input[type="tel"].six, input[type="time"].six, .row input[type="time"].six, input[type="url"].six, .row input[type="url"].six, textarea.six, .row textarea.six { width: 100% !important; }<br />
input[type="text"].seven, .row input[type="text"].seven, input[type="password"].seven, .row input[type="password"].seven, input[type="date"].seven, .row input[type="date"].seven, input[type="datetime"].seven, .row input[type="datetime"].seven, input[type="email"].seven, .row input[type="email"].seven, input[type="number"].seven, .row input[type="number"].seven, input[type="search"].seven, .row input[type="search"].seven, input[type="tel"].seven, .row input[type="tel"].seven, input[type="time"].seven, .row input[type="time"].seven, input[type="url"].seven, .row input[type="url"].seven, textarea.seven, .row textarea.seven { width: 100% !important; }<br />
input[type="text"].eight, .row input[type="text"].eight, input[type="password"].eight, .row input[type="password"].eight, input[type="date"].eight, .row input[type="date"].eight, input[type="datetime"].eight, .row input[type="datetime"].eight, input[type="email"].eight, .row input[type="email"].eight, input[type="number"].eight, .row input[type="number"].eight, input[type="search"].eight, .row input[type="search"].eight, input[type="tel"].eight, .row input[type="tel"].eight, input[type="time"].eight, .row input[type="time"].eight, input[type="url"].eight, .row input[type="url"].eight, textarea.eight, .row textarea.eight { width: 100% !important; }<br />
input[type="text"].nine, .row input[type="text"].nine, input[type="password"].nine, .row input[type="password"].nine, input[type="date"].nine, .row input[type="date"].nine, input[type="datetime"].nine, .row input[type="datetime"].nine, input[type="email"].nine, .row input[type="email"].nine, input[type="number"].nine, .row input[type="number"].nine, input[type="search"].nine, .row input[type="search"].nine, input[type="tel"].nine, .row input[type="tel"].nine, input[type="time"].nine, .row input[type="time"].nine, input[type="url"].nine, .row input[type="url"].nine, textarea.nine, .row textarea.nine { width: 100% !important; }<br />
input[type="text"].ten, .row input[type="text"].ten, input[type="password"].ten, .row input[type="password"].ten, input[type="date"].ten, .row input[type="date"].ten, input[type="datetime"].ten, .row input[type="datetime"].ten, input[type="email"].ten, .row input[type="email"].ten, input[type="number"].ten, .row input[type="number"].ten, input[type="search"].ten, .row input[type="search"].ten, input[type="tel"].ten, .row input[type="tel"].ten, input[type="time"].ten, .row input[type="time"].ten, input[type="url"].ten, .row input[type="url"].ten, textarea.ten, .row textarea.ten { width: 100% !important; }<br />
input[type="text"].eleven, .row input[type="text"].eleven, input[type="password"].eleven, .row input[type="password"].eleven, input[type="date"].eleven, .row input[type="date"].eleven, input[type="datetime"].eleven, .row input[type="datetime"].eleven, input[type="email"].eleven, .row input[type="email"].eleven, input[type="number"].eleven, .row input[type="number"].eleven, input[type="search"].eleven, .row input[type="search"].eleven, input[type="tel"].eleven, .row input[type="tel"].eleven, input[type="time"].eleven, .row input[type="time"].eleven, input[type="url"].eleven, .row input[type="url"].eleven, textarea.eleven, .row textarea.eleven { width: 100% !important; }<br />
input[type="text"].twelve, .row input[type="text"].twelve, input[type="password"].twelve, .row input[type="password"].twelve, input[type="date"].twelve, .row input[type="date"].twelve, input[type="datetime"].twelve, .row input[type="datetime"].twelve, input[type="email"].twelve, .row input[type="email"].twelve, input[type="number"].twelve, .row input[type="number"].twelve, input[type="search"].twelve, .row input[type="search"].twelve, input[type="tel"].twelve, .row input[type="tel"].twelve, input[type="time"].twelve, .row input[type="time"].twelve, input[type="url"].twelve, .row input[type="url"].twelve, textarea.twelve, .row textarea.twelve { width: 100% !important; } }<br />
/* Custom Forms ---------------------- */<br />
form.custom { /* Custom input, disabled */ }<br />
form.custom span.custom { display: inline-block; width: 16px; height: 16px; position: relative; top: 2px; border: solid 1px #ccc; background: #fff; }<br />
form.custom span.custom.radio { -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; }<br />
form.custom span.custom.checkbox:before { content: ""; display: block; line-height: 0.8; height: 14px; width: 14px; text-align: center; position: absolute; top: 0; left: 0; font-size: 14px; color: #fff; }<br />
form.custom span.custom.radio.checked:before { content: ""; display: block; width: 8px; height: 8px; -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; background: #222; position: relative; top: 3px; left: 3px; }<br />
form.custom span.custom.checkbox.checked:before { content: "\00d7"; color: #222; }<br />
form.custom div.custom.dropdown { display: block; position: relative; width: auto; height: 28px; margin-bottom: 9px; margin-top: 2px; }<br />
form.custom div.custom.dropdown a.current { display: block; width: auto; line-height: 26px; min-height: 28px; padding: 0 38px 0 6px; border: solid 1px #ddd; color: #141414; background-color: #fff; white-space: nowrap; }<br />
form.custom div.custom.dropdown a.selector { position: absolute; width: 27px; height: 28px; display: block; right: 0; top: 0; border: solid 1px #ddd; }<br />
form.custom div.custom.dropdown a.selector:after { content: ""; display: block; content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #aaaaaa transparent transparent transparent; position: absolute; left: 50%; top: 50%; margin-top: -2px; margin-left: -5px; }<br />
form.custom div.custom.dropdown:hover a.selector:after, form.custom div.custom.dropdown.open a.selector:after { content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #222222 transparent transparent transparent; }<br />
form.custom div.custom.dropdown.open ul { display: block; z-index: 10; }<br />
form.custom div.custom.dropdown.small { width: 134px !important; }<br />
form.custom div.custom.dropdown.medium { width: 254px !important; }<br />
form.custom div.custom.dropdown.large { width: 434px !important; }<br />
form.custom div.custom.dropdown.expand { width: 100% !important; }<br />
form.custom div.custom.dropdown.open.small ul { width: 134px !important; }<br />
form.custom div.custom.dropdown.open.medium ul { width: 254px !important; }<br />
form.custom div.custom.dropdown.open.large ul { width: 434px !important; }<br />
form.custom div.custom.dropdown.open.expand ul { width: 100% !important; }<br />
form.custom div.custom.dropdown ul { position: absolute; width: auto; display: none; margin: 0; left: 0; top: 27px; margin: 0; padding: 0; background: #fff; background: rgba(255, 255, 255, 0.95); border: solid 1px #cccccc; }<br />
form.custom div.custom.dropdown ul li { color: #555; font-size: 13px; cursor: pointer; padding: 3px 38px 3px 6px; min-height: 18px; line-height: 18px; margin: 0; white-space: nowrap; list-style: none; }<br />
form.custom div.custom.dropdown ul li.selected { background: #cdebf5; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:after { content: "\2013"; position: absolute; right: 10px; }<br />
form.custom div.custom.dropdown ul li:hover { background-color: #e3f4f9; color: #222; }<br />
form.custom div.custom.dropdown ul li:hover:after { content: "\2013"; position: absolute; right: 10px; color: #8ed3e7; }<br />
form.custom div.custom.dropdown ul li.selected:hover { background: #cdebf5; cursor: default; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:hover:after { color: #000; }<br />
form.custom div.custom.dropdown ul.show { display: block; }<br />
form.custom .custom.disabled { background-color: #ddd; }<br />
<br />
/* Correct FF custom dropdown height */<br />
@-moz-document url-prefix() { form.custom div.custom.dropdown a.selector { height: 30px; } }<br />
<br />
.lt-ie9 form.custom div.custom.dropdown a.selector { height: 30px; }<br />
<br />
/* Requires globals.css */<br />
/* Standard Forms ---------------------- */<br />
form { margin: 0 0 19.41641px; }<br />
<br />
.row form .row { margin: 0 -6px; }<br />
.row form .row .column, .row form .row .columns { padding: 0 6px; }<br />
.row form .row.collapse { margin: 0; }<br />
.row form .row.collapse .column, .row form .row.collapse .columns { padding: 0; }<br />
<br />
label { font-size: 14px; color: #4d4d4d; cursor: pointer; display: block; font-weight: 500; margin-bottom: 3px; }<br />
label.right { float: none; text-align: right; }<br />
label.inline { line-height: 32px; margin: 0 0 12px 0; }<br />
<br />
@media only screen and (max-width: 767px) { label.right { text-align: left; } }<br />
.prefix, .postfix { display: block; position: relative; z-index: 2; text-align: center; width: 100%; padding-top: 0; padding-bottom: 0; height: 32px; line-height: 31px; }<br />
<br />
a.button.prefix, a.button.postfix { padding-left: 0; padding-right: 0; text-align: center; }<br />
<br />
span.prefix, span.postfix { background: #f2f2f2; border: 1px solid #cccccc; }<br />
<br />
.prefix { left: 2px; -moz-border-radius-topleft: 2px; -webkit-border-top-left-radius: 2px; border-top-left-radius: 2px; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; }<br />
<br />
.postfix { right: 2px; -moz-border-radius-topright: 2px; -webkit-border-top-right-radius: 2px; border-top-right-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
input[type="text"], input[type="password"], input[type="date"], input[type="datetime"], input[type="email"], input[type="number"], input[type="search"], input[type="tel"], input[type="time"], input[type="url"], textarea { border: 1px solid #cccccc; -webkit-border-radius: 2px; -moz-border-radius: 2px; -ms-border-radius: 2px; -o-border-radius: 2px; border-radius: 2px; -webkit-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); -moz-box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); box-shadow: inset 0 1px 2px rgba(0, 0, 0, 0.1); color: rgba(0, 0, 0, 0.75); display: block; font-size: 14px; margin: 0 0 12px 0; padding: 6px; height: 32px; width: 100%; -webkit-transition: all 0.15s linear; -moz-transition: all 0.15s linear; -o-transition: all 0.15s linear; transition: all 0.15s linear; }<br />
input[type="text"].oversize, input[type="password"].oversize, input[type="date"].oversize, input[type="datetime"].oversize, input[type="email"].oversize, input[type="number"].oversize, input[type="search"].oversize, input[type="tel"].oversize, input[type="time"].oversize, input[type="url"].oversize, textarea.oversize { font-size: 17px; padding: 4px 6px; }<br />
input[type="text"]:focus, input[type="password"]:focus, input[type="date"]:focus, input[type="datetime"]:focus, input[type="email"]:focus, input[type="number"]:focus, input[type="search"]:focus, input[type="tel"]:focus, input[type="time"]:focus, input[type="url"]:focus, textarea:focus { background: #fafafa; outline: none !important; border-color: #b3b3b3; }<br />
input[type="text"][disabled], input[type="password"][disabled], input[type="date"][disabled], input[type="datetime"][disabled], input[type="email"][disabled], input[type="number"][disabled], input[type="search"][disabled], input[type="tel"][disabled], input[type="time"][disabled], input[type="url"][disabled], textarea[disabled] { background-color: #ddd; }<br />
<br />
textarea { height: auto; }<br />
<br />
select { width: 100%; }<br />
<br />
/* Fieldsets */<br />
fieldset { border: solid 1px #ddd; border-radius: 3px; -webkit-border-radius: 3px; -moz-border-radius: 3px; padding: 12px 12px 0; margin: 18px 0; }<br />
fieldset legend { font-weight: bold; background: white; padding: 0 3px; margin: 0 0 0 -3px; }<br />
<br />
/* Errors */<br />
.error input, input.error, .error textarea, textarea.error { border-color: #c60f13; background-color: rgba(198, 15, 19, 0.1); }<br />
<br />
.error label, label.error { color: #c60f13; }<br />
<br />
.error small, small.error { display: block; padding: 6px 4px; margin-top: -13px; margin-bottom: 12px; background: #c60f13; color: #fff; font-size: 12px; font-size: 1.2rem; font-weight: bold; -moz-border-radius-bottomleft: 2px; -webkit-border-bottom-left-radius: 2px; border-bottom-left-radius: 2px; -moz-border-radius-bottomright: 2px; -webkit-border-bottom-right-radius: 2px; border-bottom-right-radius: 2px; }<br />
<br />
@media only screen and (max-width: 767px) { input[type="text"].one, .row input[type="text"].one, input[type="password"].one, .row input[type="password"].one, input[type="date"].one, .row input[type="date"].one, input[type="datetime"].one, .row input[type="datetime"].one, input[type="email"].one, .row input[type="email"].one, input[type="number"].one, .row input[type="number"].one, input[type="search"].one, .row input[type="search"].one, input[type="tel"].one, .row input[type="tel"].one, input[type="time"].one, .row input[type="time"].one, input[type="url"].one, .row input[type="url"].one, textarea.one, .row textarea.one { width: 100% !important; }<br />
input[type="text"].two, .row input[type="text"].two, input[type="password"].two, .row input[type="password"].two, input[type="date"].two, .row input[type="date"].two, input[type="datetime"].two, .row input[type="datetime"].two, input[type="email"].two, .row input[type="email"].two, input[type="number"].two, .row input[type="number"].two, input[type="search"].two, .row input[type="search"].two, input[type="tel"].two, .row input[type="tel"].two, input[type="time"].two, .row input[type="time"].two, input[type="url"].two, .row input[type="url"].two, textarea.two, .row textarea.two { width: 100% !important; }<br />
input[type="text"].three, .row input[type="text"].three, input[type="password"].three, .row input[type="password"].three, input[type="date"].three, .row input[type="date"].three, input[type="datetime"].three, .row input[type="datetime"].three, input[type="email"].three, .row input[type="email"].three, input[type="number"].three, .row input[type="number"].three, input[type="search"].three, .row input[type="search"].three, input[type="tel"].three, .row input[type="tel"].three, input[type="time"].three, .row input[type="time"].three, input[type="url"].three, .row input[type="url"].three, textarea.three, .row textarea.three { width: 100% !important; }<br />
input[type="text"].four, .row input[type="text"].four, input[type="password"].four, .row input[type="password"].four, input[type="date"].four, .row input[type="date"].four, input[type="datetime"].four, .row input[type="datetime"].four, input[type="email"].four, .row input[type="email"].four, input[type="number"].four, .row input[type="number"].four, input[type="search"].four, .row input[type="search"].four, input[type="tel"].four, .row input[type="tel"].four, input[type="time"].four, .row input[type="time"].four, input[type="url"].four, .row input[type="url"].four, textarea.four, .row textarea.four { width: 100% !important; }<br />
input[type="text"].five, .row input[type="text"].five, input[type="password"].five, .row input[type="password"].five, input[type="date"].five, .row input[type="date"].five, input[type="datetime"].five, .row input[type="datetime"].five, input[type="email"].five, .row input[type="email"].five, input[type="number"].five, .row input[type="number"].five, input[type="search"].five, .row input[type="search"].five, input[type="tel"].five, .row input[type="tel"].five, input[type="time"].five, .row input[type="time"].five, input[type="url"].five, .row input[type="url"].five, textarea.five, .row textarea.five { width: 100% !important; }<br />
input[type="text"].six, .row input[type="text"].six, input[type="password"].six, .row input[type="password"].six, input[type="date"].six, .row input[type="date"].six, input[type="datetime"].six, .row input[type="datetime"].six, input[type="email"].six, .row input[type="email"].six, input[type="number"].six, .row input[type="number"].six, input[type="search"].six, .row input[type="search"].six, input[type="tel"].six, .row input[type="tel"].six, input[type="time"].six, .row input[type="time"].six, input[type="url"].six, .row input[type="url"].six, textarea.six, .row textarea.six { width: 100% !important; }<br />
input[type="text"].seven, .row input[type="text"].seven, input[type="password"].seven, .row input[type="password"].seven, input[type="date"].seven, .row input[type="date"].seven, input[type="datetime"].seven, .row input[type="datetime"].seven, input[type="email"].seven, .row input[type="email"].seven, input[type="number"].seven, .row input[type="number"].seven, input[type="search"].seven, .row input[type="search"].seven, input[type="tel"].seven, .row input[type="tel"].seven, input[type="time"].seven, .row input[type="time"].seven, input[type="url"].seven, .row input[type="url"].seven, textarea.seven, .row textarea.seven { width: 100% !important; }<br />
input[type="text"].eight, .row input[type="text"].eight, input[type="password"].eight, .row input[type="password"].eight, input[type="date"].eight, .row input[type="date"].eight, input[type="datetime"].eight, .row input[type="datetime"].eight, input[type="email"].eight, .row input[type="email"].eight, input[type="number"].eight, .row input[type="number"].eight, input[type="search"].eight, .row input[type="search"].eight, input[type="tel"].eight, .row input[type="tel"].eight, input[type="time"].eight, .row input[type="time"].eight, input[type="url"].eight, .row input[type="url"].eight, textarea.eight, .row textarea.eight { width: 100% !important; }<br />
input[type="text"].nine, .row input[type="text"].nine, input[type="password"].nine, .row input[type="password"].nine, input[type="date"].nine, .row input[type="date"].nine, input[type="datetime"].nine, .row input[type="datetime"].nine, input[type="email"].nine, .row input[type="email"].nine, input[type="number"].nine, .row input[type="number"].nine, input[type="search"].nine, .row input[type="search"].nine, input[type="tel"].nine, .row input[type="tel"].nine, input[type="time"].nine, .row input[type="time"].nine, input[type="url"].nine, .row input[type="url"].nine, textarea.nine, .row textarea.nine { width: 100% !important; }<br />
input[type="text"].ten, .row input[type="text"].ten, input[type="password"].ten, .row input[type="password"].ten, input[type="date"].ten, .row input[type="date"].ten, input[type="datetime"].ten, .row input[type="datetime"].ten, input[type="email"].ten, .row input[type="email"].ten, input[type="number"].ten, .row input[type="number"].ten, input[type="search"].ten, .row input[type="search"].ten, input[type="tel"].ten, .row input[type="tel"].ten, input[type="time"].ten, .row input[type="time"].ten, input[type="url"].ten, .row input[type="url"].ten, textarea.ten, .row textarea.ten { width: 100% !important; }<br />
input[type="text"].eleven, .row input[type="text"].eleven, input[type="password"].eleven, .row input[type="password"].eleven, input[type="date"].eleven, .row input[type="date"].eleven, input[type="datetime"].eleven, .row input[type="datetime"].eleven, input[type="email"].eleven, .row input[type="email"].eleven, input[type="number"].eleven, .row input[type="number"].eleven, input[type="search"].eleven, .row input[type="search"].eleven, input[type="tel"].eleven, .row input[type="tel"].eleven, input[type="time"].eleven, .row input[type="time"].eleven, input[type="url"].eleven, .row input[type="url"].eleven, textarea.eleven, .row textarea.eleven { width: 100% !important; }<br />
input[type="text"].twelve, .row input[type="text"].twelve, input[type="password"].twelve, .row input[type="password"].twelve, input[type="date"].twelve, .row input[type="date"].twelve, input[type="datetime"].twelve, .row input[type="datetime"].twelve, input[type="email"].twelve, .row input[type="email"].twelve, input[type="number"].twelve, .row input[type="number"].twelve, input[type="search"].twelve, .row input[type="search"].twelve, input[type="tel"].twelve, .row input[type="tel"].twelve, input[type="time"].twelve, .row input[type="time"].twelve, input[type="url"].twelve, .row input[type="url"].twelve, textarea.twelve, .row textarea.twelve { width: 100% !important; } }<br />
/* Custom Forms ---------------------- */<br />
form.custom { /* Custom input, disabled */ }<br />
form.custom span.custom { display: inline-block; width: 16px; height: 16px; position: relative; top: 2px; border: solid 1px #ccc; background: #fff; }<br />
form.custom span.custom.radio { -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; }<br />
form.custom span.custom.checkbox:before { content: ""; display: block; line-height: 0.8; height: 14px; width: 14px; text-align: center; position: absolute; top: 0; left: 0; font-size: 14px; color: #fff; }<br />
form.custom span.custom.radio.checked:before { content: ""; display: block; width: 8px; height: 8px; -webkit-border-radius: 100px; -moz-border-radius: 100px; -ms-border-radius: 100px; -o-border-radius: 100px; border-radius: 100px; background: #222; position: relative; top: 3px; left: 3px; }<br />
form.custom span.custom.checkbox.checked:before { content: "\00d7"; color: #222; }<br />
form.custom div.custom.dropdown { display: block; position: relative; width: auto; height: 28px; margin-bottom: 9px; margin-top: 2px; }<br />
form.custom div.custom.dropdown a.current { display: block; width: auto; line-height: 26px; min-height: 28px; padding: 0 38px 0 6px; border: solid 1px #ddd; color: #141414; background-color: #fff; white-space: nowrap; }<br />
form.custom div.custom.dropdown a.selector { position: absolute; width: 27px; height: 28px; display: block; right: 0; top: 0; border: solid 1px #ddd; }<br />
form.custom div.custom.dropdown a.selector:after { content: ""; display: block; content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #aaaaaa transparent transparent transparent; position: absolute; left: 50%; top: 50%; margin-top: -2px; margin-left: -5px; }<br />
form.custom div.custom.dropdown:hover a.selector:after, form.custom div.custom.dropdown.open a.selector:after { content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: #222222 transparent transparent transparent; }<br />
form.custom div.custom.dropdown.open ul { display: block; z-index: 10; }<br />
form.custom div.custom.dropdown.small { width: 134px !important; }<br />
form.custom div.custom.dropdown.medium { width: 254px !important; }<br />
form.custom div.custom.dropdown.large { width: 434px !important; }<br />
form.custom div.custom.dropdown.expand { width: 100% !important; }<br />
form.custom div.custom.dropdown.open.small ul { width: 134px !important; }<br />
form.custom div.custom.dropdown.open.medium ul { width: 254px !important; }<br />
form.custom div.custom.dropdown.open.large ul { width: 434px !important; }<br />
form.custom div.custom.dropdown.open.expand ul { width: 100% !important; }<br />
form.custom div.custom.dropdown ul { position: absolute; width: auto; display: none; margin: 0; left: 0; top: 27px; margin: 0; padding: 0; background: #fff; background: rgba(255, 255, 255, 0.95); border: solid 1px #cccccc; }<br />
form.custom div.custom.dropdown ul li { color: #555; font-size: 13px; cursor: pointer; padding: 3px 38px 3px 6px; min-height: 18px; line-height: 18px; margin: 0; white-space: nowrap; list-style: none; }<br />
form.custom div.custom.dropdown ul li.selected { background: #cdebf5; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:after { content: "\2013"; position: absolute; right: 10px; }<br />
form.custom div.custom.dropdown ul li:hover { background-color: #e3f4f9; color: #222; }<br />
form.custom div.custom.dropdown ul li:hover:after { content: "\2013"; position: absolute; right: 10px; color: #8ed3e7; }<br />
form.custom div.custom.dropdown ul li.selected:hover { background: #cdebf5; cursor: default; color: #000; }<br />
form.custom div.custom.dropdown ul li.selected:hover:after { color: #000; }<br />
form.custom div.custom.dropdown ul.show { display: block; }<br />
form.custom .custom.disabled { background-color: #ddd; }<br />
<br />
/* Correct FF custom dropdown height */<br />
@-moz-document url-prefix() { form.custom div.custom.dropdown a.selector { height: 30px; } }<br />
<br />
.lt-ie9 form.custom div.custom.dropdown a.selector { height: 30px; }<br />
<br />
/* Base Type Styles Using Modular Scale ---------------------- */<br />
body, div, dl, dt, dd, ul, ol, li, h1, h2, h3, h4, h5, h6, pre, form, fieldset, p, blockquote, th, td { margin: 0; padding: 0; font-size: 14px; }<br />
<br />
ul.orbit-bullets { margin-bottom: 17px; }<br />
<br />
p { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-weight: normal; font-size: 14px; line-height: 1.6; margin-bottom: 17px; }<br />
p.lead { font-size: 17.5px; line-height: 1.6; margin-bottom: 17px; }<br />
<br />
aside p { font-size: 13px; line-height: 1.35; font-style: italic; }<br />
<br />
h1, h2, h3, h4, h5, h6 { font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; text-rendering: optimizeLegibility; line-height: 1.1; margin-bottom: 14px; margin-top: 14px; }<br />
h1 small, h2 small, h3 small, h4 small, h5 small, h6 small { font-size: 60%; color: #6f6f6f; line-height: 0; }<br />
<br />
h1 { font-size: 44px; }<br />
<br />
h2 { font-size: 37px; }<br />
<br />
h3 { font-size: 27px; }<br />
<br />
h4 { font-size: 23px; }<br />
<br />
h5 { font-size: 17px; }<br />
<br />
h6 { font-size: 14px; }<br />
<br />
hr { border: solid #ddd; border-width: 1px 0 0; clear: both; margin: 22px 0 21px; height: 0; }<br />
<br />
.subheader { line-height: 1.3; color: #6f6f6f; font-weight: 300; margin-bottom: 17px; }<br />
<br />
em, i { font-style: italic; line-height: inherit; }<br />
<br />
strong, b { font-weight: bold; line-height: inherit; }<br />
<br />
small { font-size: 60%; line-height: inherit; }<br />
<br />
code { font-weight: bold; background: #ffff99; }<br />
<br />
/* The Grid ---------------------- */<br />
.row { width: 940px; max-width: 100%; min-width: 768px; margin: 0 auto; }<br />
.row .row { width: auto; max-width: none; min-width: 0; margin: 0 -15px; }<br />
.row.collapse .column, .row.collapse .columns { padding: 0; }<br />
.row .row { width: auto; max-width: none; min-width: 0; margin: 0 -15px; }<br />
.row .row.collapse { margin: 0; }<br />
<br />
.column, .columns { float: left; min-height: 1px; padding: 0 15px; position: relative; }<br />
.column.centered, .columns.centered { float: none; margin: 0 auto; }<br />
<br />
[class*="column"] + [class*="column"]:last-child { float: right; }<br />
<br />
[class*="column"] + [class*="column"].end { float: left; }<br />
<br />
.one, .row .one { width: 8.33333%; }<br />
<br />
.two, .row .two { width: 16.66667%; }<br />
<br />
.three, .row .three { width: 25%; }<br />
<br />
.four, .row .four { width: 33.33333%; }<br />
<br />
.five, .row .five { width: 41.66667%; }<br />
<br />
.six, .row .six { width: 50%; }<br />
<br />
.seven, .row .seven { width: 58.33333%; }<br />
<br />
.eight, .row .eight { width: 66.66667%; }<br />
<br />
.nine, .row .nine { width: 75%; }<br />
<br />
.ten, .row .ten { width: 83.33333%; }<br />
<br />
.eleven, .row .eleven { width: 91.66667%; }<br />
<br />
.twelve, .row .twelve { width: 100%; }<br />
<br />
.row .offset-by-one { margin-left: 8.33333%; }<br />
<br />
.row .offset-by-two { margin-left: 16.66667%; }<br />
<br />
.row .offset-by-three { margin-left: 25%; }<br />
<br />
.row .offset-by-four { margin-left: 33.33333%; }<br />
<br />
.row .offset-by-five { margin-left: 41.66667%; }<br />
<br />
.row .offset-by-six { margin-left: 50%; }<br />
<br />
.row .offset-by-seven { margin-left: 58.33333%; }<br />
<br />
.row .offset-by-eight { margin-left: 66.66667%; }<br />
<br />
.row .offset-by-nine { margin-left: 75%; }<br />
<br />
.row .offset-by-ten { margin-left: 83.33333%; }<br />
<br />
.push-two { left: 16.66667%; }<br />
<br />
.pull-two { right: 16.66667%; }<br />
<br />
.push-three { left: 25%; }<br />
<br />
.pull-three { right: 25%; }<br />
<br />
.push-four { left: 33.33333%; }<br />
<br />
.pull-four { right: 33.33333%; }<br />
<br />
.push-five { left: 41.66667%; }<br />
<br />
.pull-five { right: 41.66667%; }<br />
<br />
.push-six { left: 50%; }<br />
<br />
.pull-six { right: 50%; }<br />
<br />
.push-seven { left: 58.33333%; }<br />
<br />
.pull-seven { right: 58.33333%; }<br />
<br />
.push-eight { left: 66.66667%; }<br />
<br />
.pull-eight { right: 66.66667%; }<br />
<br />
.push-nine { left: 75%; }<br />
<br />
.pull-nine { right: 75%; }<br />
<br />
.push-ten { left: 83.33333%; }<br />
<br />
.pull-ten { right: 83.33333%; }<br />
<br />
img, object, embed { max-width: 100%; height: auto; }<br />
<br />
object, embed { height: 100%; }<br />
<br />
img { -ms-interpolation-mode: bicubic; }<br />
<br />
#map_canvas img, .map_canvas img { max-width: none!important; }<br />
<br />
/* Nicolas Gallagher's micro clearfix */<br />
.row { *zoom: 1; }<br />
.row:before, .row:after { content: ""; display: table; }<br />
.row:after { clear: both; }<br />
<br />
/* Mobile Grid and Overrides ---------------------- */<br />
@media only screen and (max-width: 767px) { body { -webkit-text-size-adjust: none; -ms-text-size-adjust: none; width: 100%; min-width: 0; margin-left: 0; margin-right: 0; padding-left: 0; padding-right: 0; }<br />
.row { width: auto; min-width: 0; margin-left: 0; margin-right: 0; }<br />
.column, .columns { width: auto !important; float: none; }<br />
.column:last-child, .columns:last-child { float: none; }<br />
[class*="column"] + [class*="column"]:last-child { float: none; }<br />
.column:before, .columns:before, .column:after, .columns:after { content: ""; display: table; }<br />
.column:after, .columns:after { clear: both; }<br />
.offset-by-one, .offset-by-two, .offset-by-three, .offset-by-four, .offset-by-five, .offset-by-six, .offset-by-seven, .offset-by-eight, .offset-by-nine, .offset-by-ten { margin-left: 0 !important; }<br />
.push-two, .push-three, .push-four, .push-five, .push-six, .push-seven, .push-eight, .push-nine, .push-ten { left: auto; }<br />
.pull-two, .pull-three, .pull-four, .pull-five, .pull-six, .pull-seven, .pull-eight, .pull-nine, .pull-ten { right: auto; }<br />
/* Mobile 4-column Grid */<br />
.row .mobile-one { width: 25% !important; float: left; padding: 0 15px; }<br />
.row .mobile-one:last-child { float: right; }<br />
.row.collapse .mobile-one { padding: 0; }<br />
.row .mobile-two { width: 50% !important; float: left; padding: 0 15px; }<br />
.row .mobile-two:last-child { float: right; }<br />
.row.collapse .mobile-two { padding: 0; }<br />
.row .mobile-three { width: 75% !important; float: left; padding: 0 15px; }<br />
.row .mobile-three:last-child { float: right; }<br />
.row.collapse .mobile-three { padding: 0; }<br />
.row .mobile-four { width: 100% !important; float: left; padding: 0 15px; }<br />
.row .mobile-four:last-child { float: right; }<br />
.row.collapse .mobile-four { padding: 0; }<br />
.push-one-mobile { left: 25%; }<br />
.pull-one-mobile { right: 25%; }<br />
.push-two-mobile { left: 50%; }<br />
.pull-two-mobile { right: 50%; }<br />
.push-three-mobile { left: 75%; }<br />
.pull-three-mobile { right: 75%; } }<br />
/* Block Grids ---------------------- */<br />
/* These are 2-up, 3-up, 4-up and 5-up ULs, suited<br />
for repeating blocks of content. Add 'mobile' to<br />
them to switch them just like the layout grid<br />
(one item per line) on phones<br />
<br />
For IE7/8 compatibility block-grid items need to be<br />
the same height. You can optionally uncomment the<br />
lines below to support arbitrary height, but know<br />
that IE7/8 do not support :nth-child.<br />
-------------------------------------------------- */<br />
.block-grid { display: block; overflow: hidden; padding: 0; }<br />
.block-grid > li { display: block; height: auto; float: left; }<br />
.block-grid.one-up { margin: 0; }<br />
.block-grid.one-up > li { width: 100%; padding: 0 0 15px; }<br />
.block-grid.two-up { margin: 0 -15px; }<br />
.block-grid.two-up > li { width: 50%; padding: 0 15px 15px; }<br />
.block-grid.two-up > li:nth-child(2n+1) { clear: both; }<br />
.block-grid.three-up { margin: 0 -12px; }<br />
.block-grid.three-up > li { width: 33.33%; padding: 0 12px 12px; }<br />
.block-grid.three-up > li:nth-child(3n+1) { clear: both; }<br />
.block-grid.four-up { margin: 0 -10px; }<br />
.block-grid.four-up > li { width: 25%; padding: 0 10px 10px; }<br />
.block-grid.four-up > li:nth-child(4n+1) { clear: both; }<br />
.block-grid.five-up { margin: 0 -8px; }<br />
.block-grid.five-up > li { width: 20%; padding: 0 8px 8px; }<br />
.block-grid.five-up > li:nth-child(5n+1) { clear: both; }<br />
<br />
/* Mobile Block Grids */<br />
@media only screen and (max-width: 767px) { .block-grid.mobile > li { float: none; width: 100%; margin-left: 0; }<br />
.block-grid > li { clear: none !important; }<br />
.block-grid.mobile-two-up > li { width: 50%; }<br />
.block-grid.mobile-two-up > li:nth-child(2n+1) { clear: both; }<br />
.block-grid.mobile-three-up > li { width: 33.33%; }<br />
.block-grid.mobile-three-up > li:nth-child(3n+1) { clear: both !important; }<br />
.block-grid.mobile-four-up > li { width: 25%; }<br />
.block-grid.mobile-four-up > li:nth-child(4n+1) { clear: both; }<br />
.block-grid.mobile-five-up > li:nth-child(5n+1) { clear: both; } }<br />
/* Requires globals.css */<br />
/* Normal Buttons ---------------------- */<br />
.button { width: auto; background: #2ba6cb; border: 1px solid #1e728c; -webkit-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; color: white; cursor: pointer; display: inline-block; font-family: "Helvetica Neue", "Helvetica", Helvetica, Arial, sans-serif; font-size: 14px; font-weight: bold; line-height: 1; margin: 0; outline: none; padding: 10px 20px 11px; position: relative; text-align: center; text-decoration: none; -webkit-transition: background-color 0.15s ease-in-out; -moz-transition: background-color 0.15s ease-in-out; -o-transition: background-color 0.15s ease-in-out; transition: background-color 0.15s ease-in-out; /* Hovers */ /* Sizes */ /* Colors */ /* Radii */ /* Layout */ /* Disabled ---------- */ }<br />
.button:hover { color: white; background-color: #2284a1; }<br />
.button:active { -webkit-box-shadow: 0 1px 0 rgba(0, 0, 0, 0.2) inset; -moz-box-shadow: 0 1px 0 rgba(0, 0, 0, 0.2) inset; box-shadow: 0 1px 0 rgba(0, 0, 0, 0.2) inset; }<br />
.button:focus { -webkit-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; color: white; }<br />
.button.large { font-size: 17px; padding: 15px 30px 16px; }<br />
.button.medium { font-size: 14px; }<br />
.button.small { font-size: 11px; padding: 7px 14px 8px; }<br />
.button.tiny { font-size: 10px; padding: 5px 10px 6px; }<br />
.button.expand { width: 100%; text-align: center; }<br />
.button.primary { background-color: #2ba6cb; border: 1px solid #1e728c; }<br />
.button.primary:hover { background-color: #2284a1; }<br />
.button.primary:focus { -webkit-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 4px #2ba6cb, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.success { background-color: #5da423; border: 1px solid #396516; }<br />
.button.success:hover { background-color: #457a1a; }<br />
.button.success:focus { -webkit-box-shadow: 0 0 5px #5da423, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 5px #5da423, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 5px #5da423, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.alert { background-color: #c60f13; border: 1px solid #7f0a0c; }<br />
.button.alert:hover { background-color: #970b0e; }<br />
.button.alert:focus { -webkit-box-shadow: 0 0 4px #c60f13, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 4px #c60f13, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 4px #c60f13, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.secondary { background-color: #e9e9e9; color: #1d1d1d; border: 1px solid #c3c3c3; }<br />
.button.secondary:hover { background-color: #d0d0d0; }<br />
.button.secondary:focus { -webkit-box-shadow: 0 0 5px #e9e9e9, 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 0 5px #e9e9e9, 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 0 5px #e9e9e9, 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
.button.radius { -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
.button.round { -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; }<br />
.button.full-width { width: 100%; text-align: center; padding-left: 0px !important; padding-right: 0px !important; }<br />
.button.left-align { text-align: left; text-indent: 12px; }<br />
.button.disabled, .button[disabled] { opacity: 0.6; cursor: default; background: #2ba6cb; -webkit-box-shadow: none; -moz-box-shadow: none; box-shadow: none; }<br />
.button.disabled :hover, .button[disabled] :hover { background: #2ba6cb; }<br />
.button.disabled.success, .button[disabled].success { background-color: #5da423; }<br />
.button.disabled.success:hover, .button[disabled].success:hover { background-color: #5da423; }<br />
.button.disabled.alert, .button[disabled].alert { background-color: #c60f13; }<br />
.button.disabled.alert:hover, .button[disabled].alert:hover { background-color: #c60f13; }<br />
.button.disabled.secondary, .button[disabled].secondary { background-color: #e9e9e9; }<br />
.button.disabled.secondary:hover, .button[disabled].secondary:hover { background-color: #e9e9e9; }<br />
<br />
/* Don't use native buttons on iOS */<br />
input[type=submit].button, button.button { -webkit-appearance: none; }<br />
<br />
@media only screen and (max-width: 767px) { .button { display: block; }<br />
button.button, input[type="submit"].button { width: 100%; padding-left: 0; padding-right: 0; } }<br />
/* Correct FF button padding */<br />
@-moz-document url-prefix() { button::-moz-focus-inner, input[type="reset"]::-moz-focus-inner, input[type="button"]::-moz-focus-inner, input[type="submit"]::-moz-focus-inner, input[type="file"] > input[type="button"]::-moz-focus-inner { border: none; padding: 0; }<br />
input[type="submit"].tiny.button { padding: 3px 10px 4px; }<br />
input[type="submit"].small.button { padding: 5px 14px 6px; }<br />
input[type="submit"].button, input[type=submit].medium.button { padding: 8px 20px 9px; }<br />
input[type="submit"].large.button { padding: 13px 30px 14px; } }<br />
<br />
/* Buttons with Dropdowns ---------------------- */<br />
.button.dropdown { position: relative; padding-right: 44px; /* Sizes */ /* Triangles */ /* Flyout List */ /* Split Dropdown Buttons */ }<br />
.button.dropdown.large { padding-right: 60px; }<br />
.button.dropdown.small { padding-right: 28px; }<br />
.button.dropdown.tiny { padding-right: 20px; }<br />
.button.dropdown:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: white transparent transparent transparent; position: absolute; top: 50%; right: 20px; margin-top: -2px; }<br />
.button.dropdown.large:after { content: ""; display: block; width: 0; height: 0; border: solid 7px; border-color: white transparent transparent transparent; margin-top: -3px; right: 30px; }<br />
.button.dropdown.small:after { content: ""; display: block; width: 0; height: 0; border: solid 5px; border-color: white transparent transparent transparent; margin-top: -2px; right: 14px; }<br />
.button.dropdown.tiny:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: white transparent transparent transparent; margin-top: -1px; right: 10px; }<br />
.button.dropdown > ul { -webkit-box-sizing: content-box; -moz-box-sizing: content-box; box-sizing: content-box; display: none; position: absolute; left: -1px; background: #fff; background: rgba(255, 255, 255, 0.95); list-style: none; margin: 0; padding: 0; border: 1px solid #cccccc; border-top: none; min-width: 100%; z-index: 40; }<br />
.button.dropdown > ul li { width: 100%; cursor: pointer; padding: 0; min-height: 18px; line-height: 18px; margin: 0; white-space: nowrap; list-style: none; }<br />
.button.dropdown > ul li a { display: block; color: #555; font-size: 13px; font-weight: normal; padding: 6px 14px; text-align: left; }<br />
.button.dropdown > ul li:hover { background-color: #e3f4f9; color: #222; }<br />
.button.dropdown > ul li.divider { min-height: 0; padding: 0; height: 1px; margin: 4px 0; background: #ededed; }<br />
.button.dropdown.up > ul { border-top: 1px solid #cccccc; border-bottom: none; }<br />
.button.dropdown ul.no-hover.show-dropdown { display: block !important; }<br />
.button.dropdown:hover > ul.no-hover { display: none; }<br />
.button.dropdown.split { padding: 0; position: relative; /* Sizes */ /* Triangle Spans */ /* Colors */ }<br />
.button.dropdown.split:after { display: none; }<br />
.button.dropdown.split:hover { background-color: #2ba6cb; }<br />
.button.dropdown.split.alert:hover { background-color: #c60f13; }<br />
.button.dropdown.split.success:hover { background-color: #5da423; }<br />
.button.dropdown.split.secondary:hover { background-color: #e9e9e9; }<br />
.button.dropdown.split > a { color: white; display: block; padding: 10px 50px 11px 20px; -webkit-transition: background-color 0.15s ease-in-out; -moz-transition: background-color 0.15s ease-in-out; -o-transition: background-color 0.15s ease-in-out; transition: background-color 0.15s ease-in-out; }<br />
.button.dropdown.split > a:hover { background-color: #2284a1; }<br />
.button.dropdown.split.large > a { padding: 15px 75px 16px 30px; }<br />
.button.dropdown.split.small > a { padding: 7px 35px 8px 14px; }<br />
.button.dropdown.split.tiny > a { padding: 5px 25px 6px 10px; }<br />
.button.dropdown.split > span { background-color: #2ba6cb; position: absolute; right: 0; top: 0; height: 100%; width: 30px; border-left: 1px solid #1e728c; -webkit-box-shadow: 1px 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 1px 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 1px 1px 0 rgba(255, 255, 255, 0.5) inset; -webkit-transition: background-color 0.15s ease-in-out; -moz-transition: background-color 0.15s ease-in-out; -o-transition: background-color 0.15s ease-in-out; transition: background-color 0.15s ease-in-out; }<br />
.button.dropdown.split > span:hover { background-color: #2284a1; }<br />
.button.dropdown.split > span:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: white transparent transparent transparent; position: absolute; top: 50%; left: 50%; margin-left: -6px; margin-top: -2px; }<br />
.button.dropdown.split.secondary > span:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: #1d1d1d transparent transparent transparent; }<br />
.button.dropdown.split.large span { width: 45px; }<br />
.button.dropdown.split.small span { width: 21px; }<br />
.button.dropdown.split.tiny span { width: 15px; }<br />
.button.dropdown.split.large span:after { content: ""; display: block; width: 0; height: 0; border: solid 7px; border-color: white transparent transparent transparent; margin-top: -3px; margin-left: -7px; }<br />
.button.dropdown.split.small span:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: white transparent transparent transparent; margin-top: -1px; margin-left: -4px; }<br />
.button.dropdown.split.tiny span:after { content: ""; display: block; width: 0; height: 0; border: solid 3px; border-color: white transparent transparent transparent; margin-top: -1px; margin-left: -3px; }<br />
.button.dropdown.split.alert > span { background-color: #c60f13; border-left-color: #7f0a0c; }<br />
.button.dropdown.split.success > span { background-color: #5da423; border-left-color: #396516; }<br />
.button.dropdown.split.secondary > span { background-color: #e9e9e9; border-left-color: #c3c3c3; }<br />
.button.dropdown.split.secondary > a { color: #1d1d1d; }<br />
.button.dropdown.split.alert > a:hover, .button.dropdown.split.alert > span:hover { background-color: #970b0e; }<br />
.button.dropdown.split.success > a:hover, .button.dropdown.split.success > span:hover { background-color: #457a1a; }<br />
.button.dropdown.split.secondary > a:hover, .button.dropdown.split.secondary > span:hover { background-color: #d0d0d0; }<br />
<br />
/* Button Groups ---------------------- */<br />
ul.button-group { list-style: none; padding: 0; margin: 0 0 12px; *zoom: 1; }<br />
ul.button-group:before, ul.button-group:after { content: ""; display: table; }<br />
ul.button-group:after { clear: both; }<br />
ul.button-group li { padding: 0; margin: 0 0 0 -1px; float: left; }<br />
ul.button-group li:first-child { margin-left: 0; }<br />
ul.button-group.radius li a.button, ul.button-group.radius li a.button.radius, ul.button-group.radius li a.button-rounded { -webkit-border-radius: 0px; -moz-border-radius: 0px; -ms-border-radius: 0px; -o-border-radius: 0px; border-radius: 0px; }<br />
ul.button-group.radius li:first-child a.button, ul.button-group.radius li:first-child a.button.radius { -moz-border-radius-topleft: 3px; -webkit-border-top-left-radius: 3px; border-top-left-radius: 3px; -moz-border-radius-bottomleft: 3px; -webkit-border-bottom-left-radius: 3px; border-bottom-left-radius: 3px; }<br />
ul.button-group.radius li:first-child a.button.rounded { -moz-border-radius-topleft: 1000px; -webkit-border-top-left-radius: 1000px; border-top-left-radius: 1000px; -moz-border-radius-bottomleft: 1000px; -webkit-border-bottom-left-radius: 1000px; border-bottom-left-radius: 1000px; }<br />
ul.button-group.radius li:last-child a.button, ul.button-group.radius li:last-child a.button.radius { -moz-border-radius-topright: 3px; -webkit-border-top-right-radius: 3px; border-top-right-radius: 3px; -moz-border-radius-bottomright: 3px; -webkit-border-bottom-right-radius: 3px; border-bottom-right-radius: 3px; }<br />
ul.button-group.radius li:last-child a.button.rounded { -moz-border-radius-topright: 1000px; -webkit-border-top-right-radius: 1000px; border-top-right-radius: 1000px; -moz-border-radius-bottomright: 1000px; -webkit-border-bottom-right-radius: 1000px; border-bottom-right-radius: 1000px; }<br />
ul.button-group.even a.button { width: 100%; }<br />
ul.button-group.even.two-up li { width: 50%; }<br />
ul.button-group.even.three-up li { width: 33.3%; }<br />
ul.button-group.even.three-up li:first-child { width: 33.4%; }<br />
ul.button-group.even.four-up li { width: 25%; }<br />
ul.button-group.even.five-up li { width: 20%; }<br />
<br />
@media only screen and (max-width: 767px) { .button-group button.button, .button-group input[type="submit"].button { width: auto; padding: 10px 20px 11px; }<br />
.button-group button.button.large, .button-group input[type="submit"].button.large { padding: 15px 30px 16px; }<br />
.button-group button.button.medium, .button-group input[type="submit"].button.medium { padding: 10px 20px 11px; }<br />
.button-group button.button.small, .button-group input[type="submit"].button.small { padding: 7px 14px 8px; }<br />
.button-group button.button.tiny, .button-group input[type="submit"].button.tiny { padding: 5px 10px 6px; }<br />
.button-group.even button.button, .button-group.even input[type="submit"].button { width: 100%; padding-left: 0; padding-right: 0; } }<br />
div.button-bar { overflow: hidden; }<br />
div.button-bar ul.button-group { float: left; margin-right: 8px; }<br />
div.button-bar ul.button-group:last-child { margin-left: 0; }<br />
<br />
/* Requires globals.css */<br />
.nav-bar { height: 40px; background: #4d4d4d; margin-top: 20px; padding: 0; }<br />
.nav-bar > li { float: left; display: block; position: relative; padding: 0; margin: 0; border: 1px solid #333333; border-right: none; line-height: 38px; -webkit-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset; -moz-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset; box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset; }<br />
.nav-bar > li:first-child { -webkit-box-shadow: 0 0 0; -moz-box-shadow: 0 0 0; box-shadow: 0 0 0; }<br />
.nav-bar > li:last-child { border-right: solid 1px #333333; -webkit-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset, 1px 0 0 rgba(255, 255, 255, 0.2); -moz-box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset, 1px 0 0 rgba(255, 255, 255, 0.2); box-shadow: 1px 0 0 rgba(255, 255, 255, 0.2) inset, 1px 0 0 rgba(255, 255, 255, 0.2); }<br />
.nav-bar > li.active { background: #2ba6cb; border-color: #2284a1; }<br />
.nav-bar > li.active > a { color: white; cursor: default; }<br />
.nav-bar > li.active:hover { background: #2ba6cb; cursor: default; }<br />
.nav-bar > li:hover { background: #333333; }<br />
.nav-bar > li > a { color: #e6e6e6; }<br />
.nav-bar > li ul { margin-bottom: 0; }<br />
.nav-bar > li .flyout { display: none; }<br />
.nav-bar > li.has-flyout > a:first-child { padding-right: 36px; position: relative; }<br />
.nav-bar > li.has-flyout > a:first-child:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: #e6e6e6 transparent transparent transparent; position: absolute; right: 20px; top: 17px; }<br />
.nav-bar > li.has-flyout > a.flyout-toggle { border-left: 0 !important; position: absolute; right: 0; top: 0; padding: 22px; z-index: 2; display: block; }<br />
.nav-bar > li.has-flyout.is-touch > a:first-child { padding-right: 55px; }<br />
.nav-bar > li.has-flyout.is-touch > a.flyout-toggle { border-left: 1px dashed #666; }<br />
.nav-bar > li > a:first-child { position: relative; padding: 0 20px; display: block; text-decoration: none; font-size: 14px; }<br />
.nav-bar > li > input { margin: 0 10px; }<br />
.nav-bar.vertical { height: auto; margin-top: 0; }<br />
.nav-bar.vertical > li { float: none; border-bottom: none; }<br />
.nav-bar.vertical > li.has-flyout > a:first-child:after { content: ""; display: block; width: 0; height: 0; border: solid 4px; border-color: transparent transparent transparent #e6e6e6; }<br />
.nav-bar.vertical > li .flyout { left: 100%; top: -1px; }<br />
.nav-bar.vertical > li .flyout.right { left: auto; right: 100%; }<br />
<br />
.flyout { background: #f2f2f2; padding: 20px; margin: 0; border: 1px solid #d9d9d9; position: absolute; top: 39px; left: -1px; width: 250px; z-index: 40; -webkit-box-shadow: 0 1px 5px rgba(0, 0, 0, 0.1); -moz-box-shadow: 0 1px 5px rgba(0, 0, 0, 0.1); box-shadow: 0 1px 5px rgba(0, 0, 0, 0.1); /* remove margin on any first-child element */ /* remove margin on last element */ }<br />
.flyout p { line-height: 1.2; font-size: 13px; }<br />
.flyout *:first-child { margin-top: 0; }<br />
.flyout *:last-child { margin-bottom: 0; }<br />
.flyout.small { width: 166.66667px; }<br />
.flyout.large { width: 437.5px; }<br />
.flyout.right { left: auto; right: -2px; }<br />
.flyout.up { top: auto; bottom: 39px; }<br />
<br />
ul.flyout, .nav-bar li ul { padding: 0; list-style: none; }<br />
ul.flyout li, .nav-bar li ul li { border-left: solid 3px #CCC; }<br />
ul.flyout li a, .nav-bar li ul li a { background: #f2f2f2; border: 1px solid #e6e6e6; border-width: 1px 1px 0 0; color: #555; display: block; font-size: 14px; height: auto; line-height: 1; padding: 15px 20px; -webkit-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
ul.flyout li a:hover, .nav-bar li ul li a:hover { background: #ebebeb; color: #333; }<br />
ul.flyout li.active, .nav-bar li ul li.active { margin-top: 0; border-top: 1px solid #4d4d4d; border-left: 4px solid #1a1a1a; }<br />
ul.flyout li.active a, .nav-bar li ul li.active a { background: #4d4d4d; border: none; color: #fff; height: auto; margin: 0; position: static; top: 0; -webkit-box-shadow: 0 0 0; -moz-box-shadow: 0 0 0; box-shadow: 0 0 0; }<br />
<br />
/* Mobile Styles */<br />
@media only screen and (max-device-width: 1280px) { .touch .nav-bar li.has-flyout > a { padding-right: 36px !important; } }<br />
@media only screen and (max-width: 1279px) and (min-width: 768px) { .touch .nav-bar li a { font-size: 13px; font-size: 1.3rem; }<br />
.touch .nav-bar li.has-flyout > a.flyout-toggle { padding: 20px !important; }<br />
.touch .nav-bar li.has-flyout > a { padding-right: 36px !important; } }<br />
@media only screen and (max-width: 767px) { .nav-bar { height: auto; }<br />
.nav-bar > li { float: none; display: block; border-right: none; }<br />
.nav-bar > li > a.main { text-align: left; border-top: 1px solid #ddd; border-right: none; }<br />
.nav-bar > li:first-child > a.main { border-top: none; }<br />
.nav-bar > li.has-flyout > a.flyout-toggle { position: absolute; right: 0; top: 0; padding: 22px; z-index: 2; display: block; }<br />
.nav-bar > li.has-flyout.is-touch > a.flyout-toggle span { content: ""; width: 0; height: 0; display: block; }<br />
.nav-bar > li.has-flyout > a.flyout-toggle:hover span { border-top-color: #141414; }<br />
.nav-bar.vertical > li.has-flyout > .flyout { left: 0; }<br />
.flyout { position: relative; width: 100% !important; top: auto; margin-right: -2px; border-width: 1px 1px 0 1px; }<br />
.flyout.right { float: none; right: auto; left: -1px; }<br />
.flyout.small, .flyout.large { width: 100% !important; }<br />
.flyout p:last-child { margin-bottom: 18px; } }<br />
/* CSS for jQuery Orbit Plugin 1.4.0 Maintained for Foundation. foundation.zurb.com Free to use under the MIT license. http://www.opensource.org/licenses/mit-license.php<br />
*/<br />
/* Container ---------------------- */<br />
div.orbit-wrapper { width: 1px; height: 1px; position: relative; }<br />
<br />
div.orbit { width: 1px; height: 1px; position: relative; overflow: hidden; margin-bottom: 17px; }<br />
<br />
div.orbit.with-bullets { margin-bottom: 40px; }<br />
<br />
div.orbit .orbit-slide { max-width: 100%; position: absolute; top: 0; left: 0; }<br />
<br />
div.orbit a.orbit-slide { border: none; line-height: 0; display: none; }<br />
<br />
div.orbit div.orbit-slide { width: 100%; height: 100%; }<br />
<br />
/* Note: If your slider only uses content or anchors, you're going to want to put the width and height declarations on the ".orbit>div" and "div.orbit>a" tags in addition to just the .orbit-wrapper */<br />
/* Timer ---------------------- */<br />
div.orbit-wrapper div.timer { width: 40px; height: 40px; overflow: hidden; position: absolute; top: 10px; right: 10px; opacity: .6; cursor: pointer; z-index: 31; }<br />
<br />
div.orbit-wrapper span.rotator { display: block; width: 40px; height: 40px; position: absolute; top: 0; left: -20px; background: url('https://static.igem.org/mediawiki/2013/7/72/CornellRotator-black.png') no-repeat; z-index: 3; }<br />
<br />
div.orbit-wrapper span.mask { display: block; width: 20px; height: 40px; position: absolute; top: 0; right: 0; z-index: 2; overflow: hidden; }<br />
<br />
div.orbit-wrapper span.rotator.move { left: 0; }<br />
<br />
div.orbit-wrapper span.mask.move { width: 40px; left: 0; background: url('https://static.igem.org/mediawiki/2013/8/8a/CornellTimer-black.png') repeat 0 0; }<br />
<br />
div.orbit-wrapper span.pause { display: block; width: 40px; height: 40px; position: absolute; top: 0; left: 0; background: url('https://static.igem.org/mediawiki/2013/e/e8/CornellPause-black.png') no-repeat; z-index: 4; opacity: 0; }<br />
<br />
div.orbit-wrapper span.pause.active { background: url('https://static.igem.org/mediawiki/2013/e/e8/CornellPause-black.png') no-repeat 0 -40px; }<br />
<br />
div.orbit-wrapper div.timer:hover span.pause, div.orbit-wrapper span.pause.active { opacity: 1; }<br />
<br />
/* Captions ---------------------- */<br />
.orbit-caption { display: none; font-family: "HelveticaNeue", "Helvetica-Neue", Helvetica, Arial, sans-serif; }<br />
<br />
.orbit-wrapper .orbit-caption { background: #000; background: rgba(0, 0, 0, 0.6); z-index: 30; color: #fff; text-align: center; padding: 7px 0; font-size: 13px; font-size: 1.3rem; position: absolute; right: 0; bottom: 0; width: 100%; }<br />
<br />
/* Directional Nav ---------------------- */<br />
div.orbit-wrapper div.slider-nav { display: block; }<br />
<br />
div.orbit-wrapper div.slider-nav span { width: 39px; height: 50px; text-indent: -9999px; position: absolute; z-index: 30; top: 50%; margin-top: -25px; cursor: pointer; }<br />
<br />
div.orbit-wrapper div.slider-nav span.right { background: url('https://static.igem.org/mediawiki/2013/3/3e/CornellRight-arrow.png'); background-size: 100%; right: 0; }<br />
<br />
div.orbit-wrapper div.slider-nav span.left { background: url('https://static.igem.org/mediawiki/2013/e/e7/Left-arrow.png'); background-size: 100%; left: 0; }<br />
<br />
.lt-ie9 div.orbit-wrapper div.slider-nav span.right { background: url('https://static.igem.org/mediawiki/2013/9/96/Right-arrow-small.png'); }<br />
.lt-ie9 div.orbit-wrapper div.slider-nav span.left { background: url('https://static.igem.org/mediawiki/2013/d/de/Left-arrow-small.png'); }<br />
<br />
/* Bullet Nav ---------------------- */<br />
ul.orbit-bullets { position: absolute; z-index: 30; list-style: none; bottom: -40px; left: 50%; margin-left: -50px; padding: 0; }<br />
<br />
ul.orbit-bullets li { float: left; margin-left: 5px; cursor: pointer; color: #999; text-indent: -9999px; background: url('https://static.igem.org/mediawiki/2013/3/3b/CornellBullets.png') no-repeat 4px 0; width: 13px; height: 12px; overflow: hidden; }<br />
<br />
ul.orbit-bullets li.active { color: #222; background-position: -8px 0; }<br />
<br />
ul.orbit-bullets li.has-thumb { background: none; width: 100px; height: 75px; }<br />
<br />
ul.orbit-bullets li.active.has-thumb { background-position: 0 0; border-top: 2px solid #000; }<br />
<br />
/* Fluid Layout ---------------------- */<br />
div.orbit img.fluid-placeholder { visibility: hidden; position: static; display: block; width: 100%; }<br />
<br />
div.orbit, div.orbit-wrapper { width: 100% !important; }<br />
<br />
ul.orbit-bullets { position: absolute; z-index: 30; list-style: none; bottom: -50px; left: 50%; margin-left: -50px; padding: 0; }<br />
<br />
ul.orbit-bullets li { float: left; margin-left: 5px; cursor: pointer; color: #999; text-indent: -9999px; background: url('https://static.igem.org/mediawiki/2013/3/3b/CornellBullets.png') no-repeat 4px 0; width: 13px; height: 12px; overflow: hidden; }<br />
<br />
ul.orbit-bullets li.has-thumb { background: none; width: 100px; height: 75px; }<br />
<br />
ul.orbit-bullets li.active { color: #222; background-position: -8px 0; }<br />
<br />
ul.orbit-bullets li.active.has-thumb { background-position: 0 0; border-top: 2px solid #000; }<br />
<br />
/* Correct timer in IE */<br />
.lt-ie9 .timer { display: none !important; }<br />
<br />
.lt-ie9 div.caption { background: transparent; filter: progid:DXImageTransform.Microsoft.gradient(startColorstr=#99000000,endColorstr=#99000000); zoom: 1; }<br />
<br />
/* CSS for jQuery Reveal Plugin Maintained for Foundation. foundation.zurb.com Free to use under the MIT license. http://www.opensource.org/licenses/mit-license.php */<br />
/* Reveal Modals ---------------------- */<br />
.reveal-modal-bg { position: fixed; height: 100%; width: 100%; background: #000; background: rgba(0, 0, 0, 0.45); z-index: 40; display: none; top: 0; left: 0; }<br />
<br />
.reveal-modal { background: white; visibility: hidden; display: none; top: 100px; left: 50%; margin-left: -260px; width: 520px; position: absolute; z-index: 41; padding: 30px; -webkit-box-shadow: 0 0 10px rgba(0, 0, 0, 0.4); -moz-box-shadow: 0 0 10px rgba(0, 0, 0, 0.4); box-shadow: 0 0 10px rgba(0, 0, 0, 0.4); }<br />
.reveal-modal *:first-child { margin-top: 0; }<br />
.reveal-modal *:last-child { margin-bottom: 0; }<br />
.reveal-modal .close-reveal-modal { font-size: 22px; font-size: 2.2rem; line-height: .5; position: absolute; top: 8px; right: 11px; color: #aaa; text-shadow: 0 -1px 1px rgba(0, 0, 0, 0.6); font-weight: bold; cursor: pointer; }<br />
.reveal-modal.small { width: 30%; margin-left: -10%; }<br />
.reveal-modal.medium { width: 40%; margin-left: -20%; }<br />
.reveal-modal.large { width: 60%; margin-left: -30%; }<br />
.reveal-modal.xlarge { width: 70%; margin-left: -35%; }<br />
.reveal-modal.expand { width: 90%; margin-left: -45%; }<br />
.reveal-modal .row { min-width: 0; }<br />
<br />
/* Mobile */<br />
@media only screen and (max-width: 767px) { .reveal-modal-bg { position: absolute; }<br />
.reveal-modal, .reveal-modal.small, .reveal-modal.medium, .reveal-modal.large, .reveal-modal.xlarge { width: 80%; top: 15px; left: 50%; margin-left: -40%; padding: 20px; height: auto; } }<br />
/* NOTES Close button entity is &#215;<br />
Example markup <div id="myModal" class="reveal-modal"> <h2>Awesome. I have it.</h2> <p class="lead">Your couch. I it's mine.</p> <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. In ultrices aliquet placerat. Duis pulvinar orci et nisi euismod vitae tempus lorem consectetur. Duis at magna quis turpis mattis venenatis eget id diam. </p> <a class="close-reveal-modal">&#215;</a> </div> */<br />
/* Requires globals.css app.js */<br />
/* Tabs ---------------------- */<br />
dl.tabs { border-bottom: solid 1px #e6e6e6; display: block; height: 40px; padding: 0; margin-bottom: 20px; }<br />
dl.tabs.contained { margin-bottom: 0; }<br />
dl.tabs dt { color: #b3b3b3; cursor: default; display: block; float: left; font-size: 12px; height: 40px; line-height: 40px; padding: 0 9px 0 20px; width: auto; text-transform: uppercase; }<br />
dl.tabs dt:first-child { padding: 0 9px 0 0; }<br />
dl.tabs dd { display: block; float: left; padding: 0; margin: 0; }<br />
dl.tabs dd a { color: #6f6f6f; display: block; font-size: 14px; height: 40px; line-height: 40px; padding: 0px 23.8px; }<br />
dl.tabs dd a:focus { font-weight: bold; color: #2ba6cb; }<br />
dl.tabs dd.active { border-top: 3px solid #2ba6cb; margin-top: -3px; }<br />
dl.tabs dd.active a { cursor: default; color: #3c3c3c; background: #fff; border-left: 1px solid #e6e6e6; border-right: 1px solid #e6e6e6; font-weight: bold; }<br />
dl.tabs dd:first-child { margin-left: 0; }<br />
dl.tabs.vertical { height: auto; border-bottom: 1px solid #e6e6e6; }<br />
dl.tabs.vertical dt, dl.tabs.vertical dd { float: none; height: auto; }<br />
dl.tabs.vertical dd { border-left: 3px solid #cccccc; }<br />
dl.tabs.vertical dd a { background: #f2f2f2; border: none; border: 1px solid #e6e6e6; border-width: 1px 1px 0 0; color: #555; display: block; font-size: 14px; height: auto; line-height: 1; padding: 15px 20px; -webkit-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; -moz-box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; box-shadow: 0 1px 0 rgba(255, 255, 255, 0.5) inset; }<br />
dl.tabs.vertical dd.active { margin-top: 0; border-top: 1px solid #4d4d4d; border-left: 4px solid #1a1a1a; }<br />
dl.tabs.vertical dd.active a { background: #4d4d4d; border: none; color: #fff; height: auto; margin: 0; position: static; top: 0; -webkit-box-shadow: 0 0 0; -moz-box-shadow: 0 0 0; box-shadow: 0 0 0; }<br />
dl.tabs.vertical dd:first-child a.active { margin: 0; }<br />
dl.tabs.pill { border-bottom: none; margin-bottom: 10px; }<br />
dl.tabs.pill dd { margin-right: 10px; }<br />
dl.tabs.pill dd:last-child { margin-right: 0; }<br />
dl.tabs.pill dd a { -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; background: #e6e6e6; height: 26px; line-height: 26px; color: #666; }<br />
dl.tabs.pill dd.active { border: none; margin-top: 0; }<br />
dl.tabs.pill dd.active a { background-color: #2ba6cb; border: none; color: #fff; }<br />
dl.tabs.pill.contained { border-bottom: solid 1px #eee; margin-bottom: 0; }<br />
dl.tabs.two-up dt a, dl.tabs.two-up dd a, dl.tabs.three-up dt a, dl.tabs.three-up dd a, dl.tabs.four-up dt a, dl.tabs.four-up dd a, dl.tabs.five-up dt a, dl.tabs.five-up dd a { padding: 0 17px; text-align: center; overflow: hidden; }<br />
dl.tabs.two-up dt, dl.tabs.two-up dd { width: 50%; }<br />
dl.tabs.three-up dt, dl.tabs.three-up dd { width: 33.33%; }<br />
dl.tabs.four-up dt, dl.tabs.four-up dd { width: 25%; }<br />
dl.tabs.five-up dt, dl.tabs.five-up dd { width: 20%; }<br />
<br />
ul.tabs-content { display: block; margin: 0 0 20px; padding: 0; }<br />
ul.tabs-content > li { display: none; }<br />
ul.tabs-content > li.active { display: block; }<br />
ul.tabs-content.contained { padding: 0; }<br />
ul.tabs-content.contained > li { border: solid 0 #e6e6e6; border-width: 0 1px 1px 1px; padding: 20px; }<br />
ul.tabs-content.contained.vertical > li { border-width: 1px 1px 1px 1px; }<br />
<br />
.no-js ul.tabs-content > li { display: block; }<br />
<br />
@media only screen and (max-width: 767px) { dl.tabs.mobile { width: auto; margin: 20px -20px 40px; height: auto; }<br />
dl.tabs.mobile dt, dl.tabs.mobile dd { float: none; height: auto; }<br />
dl.tabs.mobile dd a { display: block; width: auto; height: auto; padding: 18px 20px; line-height: 1; border: solid 0 #ccc; border-width: 1px 0 0; margin: 0; color: #555; background: #eee; font-size: 15px; font-size: 1.5rem; }<br />
dl.tabs.mobile dd a.active { height: auto; margin: 0; border-width: 1px 0 0; }<br />
.tabs.mobile { border-bottom: solid 1px #ccc; height: auto; }<br />
.tabs.mobile dd a { padding: 18px 20px; border: none; border-left: none; border-right: none; border-top: 1px solid #ccc; background: #fff; }<br />
.tabs.mobile dd a.active { border: none; background: #2ba6cb; color: #fff; margin: 0; position: static; top: 0; height: auto; }<br />
.tabs.mobile dd:first-child a.active { margin: 0; }<br />
dl.contained.mobile { margin-bottom: 0; }<br />
dl.contained.tabs.mobile dd a { padding: 18px 20px; }<br />
dl.tabs.mobile + ul.contained { margin-left: -20px; margin-right: -20px; border-width: 0 0 1px 0; } }<br />
/* Requires: globals.css */<br />
/* Table of Contents<br />
<br />
:: Visibility<br />
:: Alerts<br />
:: Labels<br />
:: Tooltips<br />
:: Panels<br />
:: Accordion<br />
:: Side Nav<br />
:: Sub Nav<br />
:: Pagination<br />
:: Breadcrumbs<br />
:: Lists<br />
:: Link Lists<br />
:: Keystroke Chars<br />
:: Video<br />
:: Tables<br />
:: Microformats<br />
<br />
*/<br />
/* Visibility Classes ---------------------- */<br />
/* Standard (large) display targeting */<br />
.show-for-small, .show-for-medium, .show-for-medium-down, .hide-for-large, .hide-for-large-up, .show-for-xlarge { display: none !important; }<br />
<br />
.hide-for-xlarge, .show-for-large, .show-for-large-up, .hide-for-small, .hide-for-medium, .hide-for-medium-down { display: block !important; }<br />
<br />
/* Very large display targeting */<br />
@media only screen and (min-width: 1441px) { .hide-for-small, .hide-for-medium, .hide-for-medium-down, .hide-for-large, .show-for-large-up, .show-for-xlarge { display: block !important; }<br />
.show-for-small, .show-for-medium, .show-for-medium-down, .show-for-large, .hide-for-large-up, .hide-for-xlarge { display: none !important; } }<br />
/* Medium display targeting */<br />
@media only screen and (max-width: 1279px) and (min-width: 768px) { .hide-for-small, .show-for-medium, .show-for-medium-down, .hide-for-large, .hide-for-large-up, .hide-for-xlarge { display: block !important; }<br />
.show-for-small, .hide-for-medium, .hide-for-medium-down, .show-for-large, .show-for-large-up, .show-for-xlarge { display: none !important; } }<br />
/* Small display targeting */<br />
@media only screen and (max-width: 767px) { .show-for-small, .hide-for-medium, .show-for-medium-down, .hide-for-large, .hide-for-large-up, .hide-for-xlarge { display: block !important; }<br />
.hide-for-small, .show-for-medium, .hide-for-medium-down, .show-for-large, .show-for-large-up, .show-for-xlarge { display: none !important; } }<br />
/* Orientation targeting */<br />
.show-for-landscape, .hide-for-portrait { display: block !important; }<br />
<br />
.hide-for-landscape, .show-for-portrait { display: none !important; }<br />
<br />
@media screen and (orientation: landscape) { .show-for-landscape, .hide-for-portrait { display: block !important; }<br />
.hide-for-landscape, .show-for-portrait { display: none !important; } }<br />
@media screen and (orientation: portrait) { .show-for-portrait, .hide-for-landscape { display: block !important; }<br />
.hide-for-portrait, .show-for-landscape { display: none !important; } }<br />
/* Touch-enabled device targeting */<br />
.show-for-touch { display: none !important; }<br />
<br />
.hide-for-touch { display: block !important; }<br />
<br />
.touch .show-for-touch { display: block !important; }<br />
<br />
.touch .hide-for-touch { display: none !important; }<br />
<br />
/* Specific overrides for elements that require something other than display: block */<br />
table.show-for-xlarge, table.show-for-large, table.hide-for-small, table.hide-for-medium { display: table !important; }<br />
<br />
@media only screen and (max-width: 1279px) and (min-width: 768px) { .touch table.hide-for-xlarge, .touch table.hide-for-large, .touch table.hide-for-small, .touch table.show-for-medium { display: table !important; } }<br />
@media only screen and (max-width: 767px) { table.hide-for-xlarge, table.hide-for-large, table.hide-for-medium, table.show-for-small { display: table !important; } }<br />
/* Alerts ---------------------- */<br />
div.alert-box { display: block; padding: 6px 7px 7px; font-weight: bold; font-size: 14px; color: white; background-color: #2ba6cb; border: 1px solid rgba(0, 0, 0, 0.1); margin-bottom: 12px; -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; text-shadow: 0 -1px rgba(0, 0, 0, 0.3); position: relative; }<br />
div.alert-box.success { background-color: #5da423; color: #fff; text-shadow: 0 -1px rgba(0, 0, 0, 0.3); }<br />
div.alert-box.alert { background-color: #c60f13; color: #fff; text-shadow: 0 -1px rgba(0, 0, 0, 0.3); }<br />
div.alert-box.secondary { background-color: #e9e9e9; color: #505050; text-shadow: 0 1px rgba(255, 255, 255, 0.3); }<br />
div.alert-box a.close { color: #333; position: absolute; right: 4px; top: -1px; font-size: 17px; opacity: 0.2; padding: 4px; }<br />
div.alert-box a.close:hover, div.alert-box a.close:focus { opacity: 0.4; }<br />
<br />
/* Labels ---------------------- */<br />
.label { padding: 1px 4px 2px; font-size: 12px; font-weight: bold; text-align: center; text-decoration: none; line-height: 1; white-space: nowrap; display: inline; position: relative; bottom: 1px; color: #fff; background: #2ba6cb; }<br />
.label.radius { -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
.label.round { padding: 1px 7px 2px; -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; }<br />
.label.alert { background-color: #c60f13; }<br />
.label.success { background-color: #5da423; }<br />
.label.secondary { background-color: #e9e9e9; color: #505050; }<br />
<br />
/* Tooltips ---------------------- */<br />
.has-tip { border-bottom: dotted 1px #cccccc; cursor: help; font-weight: bold; color: #333333; }<br />
.has-tip:hover { border-bottom: dotted 1px #196177; color: #2ba6cb; }<br />
.has-tip.tip-left, .has-tip.tip-right { float: none !important; }<br />
<br />
.tooltip { display: none; background: black; background: rgba(0, 0, 0, 0.85); position: absolute; color: white; font-weight: bold; font-size: 12px; font-size: 1.2rem; padding: 5px; z-index: 999; -webkit-border-radius: 4px; -moz-border-radius: 4px; border-radius: 4px; line-height: normal; }<br />
.tooltip > .nub { display: block; width: 0; height: 0; border: solid 5px; border-color: transparent transparent black transparent; border-color: transparent transparent rgba(0, 0, 0, 0.85) transparent; position: absolute; top: -10px; left: 10px; }<br />
.tooltip.tip-override > .nub { border-color: transparent transparent black transparent !important; border-color: transparent transparent rgba(0, 0, 0, 0.85) transparent !important; top: -10px !important; }<br />
.tooltip.tip-top > .nub { border-color: black transparent transparent transparent; border-color: rgba(0, 0, 0, 0.85) transparent transparent transparent; top: auto; bottom: -10px; }<br />
.tooltip.tip-left, .tooltip.tip-right { float: none !important; }<br />
.tooltip.tip-left > .nub { border-color: transparent transparent transparent black; border-color: transparent transparent transparent rgba(0, 0, 0, 0.85); right: -10px; left: auto; }<br />
.tooltip.tip-right > .nub { border-color: transparent black transparent transparent; border-color: transparent rgba(0, 0, 0, 0.85) transparent transparent; right: auto; left: -10px; }<br />
.tooltip.noradius { -webkit-border-radius: 0; -moz-border-radius: 0; -ms-border-radius: 0; -o-border-radius: 0; border-radius: 0; }<br />
.tooltip.opened { color: #2ba6cb !important; border-bottom: dotted 1px #196177 !important; }<br />
<br />
.tap-to-close { display: block; font-size: 10px; font-size: 1rem; color: #888888; font-weight: normal; }<br />
<br />
@media only screen and (max-width: 767px) { .tooltip { font-size: 14px; font-size: 1.4rem; line-height: 1.4; padding: 7px 10px 9px 10px; }<br />
.tooltip > .nub, .tooltip.top > .nub, .tooltip.left > .nub, .tooltip.right > .nub { border-color: transparent transparent black transparent; border-color: transparent transparent rgba(0, 0, 0, 0.85) transparent; top: -12px; left: 10px; } }<br />
/* Panels ---------------------- */<br />
.panel { background: #f2f2f2; border: solid 1px #e6e6e6; margin: 0 0 22px 0; padding: 20px; }<br />
.panel > :first-child { margin-top: 0; }<br />
.panel > :last-child { margin-bottom: 0; }<br />
.panel.callout { background: #2ba6cb; color: #fff; border-color: #2284a1; -webkit-box-shadow: inset 0px 1px 0px rgba(255, 255, 255, 0.5); -moz-box-shadow: inset 0px 1px 0px rgba(255, 255, 255, 0.5); box-shadow: inset 0px 1px 0px rgba(255, 255, 255, 0.5); }<br />
.panel.callout a { color: #fff; }<br />
.panel.callout .button { background: white; border: none; color: #2ba6cb; text-shadow: none; }<br />
.panel.callout .button:hover { background: rgba(255, 255, 255, 0.8); }<br />
.panel.radius { -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
<br />
/* Accordion ---------------------- */<br />
ul.accordion { margin: 0 0 22px 0; border-bottom: 1px solid #e9e9e9; }<br />
ul.accordion > li { list-style: none; margin: 0; padding: 0; border-top: 1px solid #e9e9e9; }<br />
ul.accordion > li .title { cursor: pointer; background: #f6f6f6; padding: 15px; margin: 0; position: relative; border-left: 1px solid #e9e9e9; border-right: 1px solid #e9e9e9; -webkit-transition: 0.15s background linear; -moz-transition: 0.15s background linear; -o-transition: 0.15s background linear; transition: 0.15s background linear; }<br />
ul.accordion > li .title h1, ul.accordion > li .title h2, ul.accordion > li .title h3, ul.accordion > li .title h4, ul.accordion > li .title h5 { margin: 0; }<br />
ul.accordion > li .title:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: transparent #9d9d9d transparent transparent; position: absolute; right: 15px; top: 21px; }<br />
ul.accordion > li .content { display: none; padding: 15px; }<br />
ul.accordion > li.active { border-top: 3px solid #2ba6cb; }<br />
ul.accordion > li.active .title { background: white; padding-top: 13px; }<br />
ul.accordion > li.active .title:after { content: ""; display: block; width: 0; height: 0; border: solid 6px; border-color: #9d9d9d transparent transparent transparent; }<br />
ul.accordion > li.active .content { background: white; display: block; border-left: 1px solid #e9e9e9; border-right: 1px solid #e9e9e9; }<br />
<br />
/* Side Nav ---------------------- */<br />
ul.side-nav { display: block; list-style: none; margin: 0; padding: 17px 0; }<br />
ul.side-nav li { display: block; list-style: none; margin: 0 0 7px 0; }<br />
ul.side-nav li a { display: block; }<br />
ul.side-nav li.active a { color: #4d4d4d; font-weight: bold; }<br />
ul.side-nav li.inactive a { color: #b31b1b; font-weight: normal; }<br />
ul.side-nav li.divider { border-top: 1px solid #e6e6e6; height: 0; padding: 0; }<br />
<br />
/* Sub Navs http://www.zurb.com/article/292/how-to-create-simple-and-effective-sub-na ---------------------- */<br />
dl.sub-nav { display: block; width: auto; overflow: hidden; margin: -4px 0 18px -9px; padding-top: 4px; }<br />
dl.sub-nav dt, dl.sub-nav dd { float: left; display: inline; margin-left: 9px; margin-bottom: 10px; }<br />
dl.sub-nav dt { color: #999; font-weight: normal; }<br />
dl.sub-nav dd a { text-decoration: none; -webkit-border-radius: 1000px; -moz-border-radius: 1000px; -ms-border-radius: 1000px; -o-border-radius: 1000px; border-radius: 1000px; }<br />
dl.sub-nav dd.active a { font-weight: bold; background: #2ba6cb; color: #fff; padding: 3px 9px; cursor: default; }<br />
<br />
/* Pagination ---------------------- */<br />
ul.pagination { display: block; height: 24px; margin-left: -5px; }<br />
ul.pagination li { float: left; display: block; height: 24px; color: #999; font-size: 14px; margin-left: 5px; }<br />
ul.pagination li a { display: block; padding: 1px 7px 1px; color: #555; }<br />
ul.pagination li:hover a, ul.pagination li a:focus { background: #e6e6e6; }<br />
ul.pagination li.unavailable a { cursor: default; color: #999; }<br />
ul.pagination li.unavailable:hover a, ul.pagination li.unavailable a:focus { background: transparent; }<br />
ul.pagination li.current a { background: #2ba6cb; color: white; font-weight: bold; cursor: default; }<br />
ul.pagination li.current a:hover { background: #2ba6cb; }<br />
<br />
/* Breadcrums ---------------------- */<br />
ul.breadcrumbs { display: block; background: #f6f6f6; padding: 6px 10px 7px; border: 1px solid #e9e9e9; -webkit-border-radius: 2px; -moz-border-radius: 2px; -ms-border-radius: 2px; -o-border-radius: 2px; border-radius: 2px; overflow: hidden; }<br />
ul.breadcrumbs li { margin: 0; padding: 0 12px 0 0; float: left; list-style: none; }<br />
ul.breadcrumbs li a, ul.breadcrumbs li span { text-transform: uppercase; font-size: 11px; font-size: 1.1rem; padding-left: 12px; }<br />
ul.breadcrumbs li:first-child a, ul.breadcrumbs li:first-child span { padding-left: 0; }<br />
ul.breadcrumbs li:before { content: "/"; color: #aaa; }<br />
ul.breadcrumbs li:first-child:before { content: " "; }<br />
ul.breadcrumbs li.current a { cursor: default; color: #333; }<br />
ul.breadcrumbs li:hover a, ul.breadcrumbs li a:focus { text-decoration: underline; }<br />
ul.breadcrumbs li.current:hover a, ul.breadcrumbs li.current a:focus { text-decoration: none; }<br />
ul.breadcrumbs li.unavailable a { color: #999; }<br />
ul.breadcrumbs li.unavailable:hover a, ul.breadcrumbs li.unavailable a:focus { text-decoration: none; color: #999; cursor: default; }<br />
<br />
/* Link List */<br />
ul.link-list { margin: 0 0 17px -22px; padding: 0; list-style: none; overflow: hidden; }<br />
ul.link-list li { list-style: none; float: left; margin-left: 22px; display: block; }<br />
ul.link-list li a { display: block; }<br />
<br />
/* Keytroke Characters ---------------------- */<br />
.keystroke, kbd { font-family: "Consolas", "Menlo", "Courier", monospace; font-size: 13px; padding: 2px 4px 0px; margin: 0; background: #ededed; border: solid 1px #dbdbdb; -webkit-border-radius: 3px; -moz-border-radius: 3px; -ms-border-radius: 3px; -o-border-radius: 3px; border-radius: 3px; }<br />
<br />
/* Video - Mad props to http://www.alistapart.com/articles/creating-intrinsic-ratios-for-video/ ---------------------- */<br />
.flex-video { position: relative; padding-top: 25px; padding-bottom: 67.5%; height: 0; margin-bottom: 16px; overflow: hidden; }<br />
.flex-video.widescreen { padding-bottom: 57.25%; }<br />
.flex-video.vimeo { padding-top: 0; }<br />
.flex-video iframe, .flex-video object, .flex-video embed, .flex-video video { position: absolute; top: 0; left: 0; width: 100%; height: 100%; }<br />
<br />
@media only screen and (max-device-width: 800px), only screen and (device-width: 1024px) and (device-height: 600px), only screen and (width: 1280px) and (orientation: landscape), only screen and (device-width: 800px), only screen and (max-width: 767px) { .flex-video { padding-top: 0; } }<br />
/* Tables ---------------------- */<br />
table { background: #fff; -moz-border-radius: 3px; -webkit-border-radius: 3px; border-radius: 3px; margin: 0 0 18px; border: 1px solid #ddd; }<br />
<br />
table thead, table tfoot { background: #f5f5f5; }<br />
<br />
table thead tr th, table tfoot tr th, table tbody tr td, table tr td, table tfoot tr td { font-size: 14px; line-height: 18px; text-align: left; }<br />
<br />
table thead tr th, table tfoot tr td { padding: 8px 10px 9px; font-size: 16px; font-weight: bold; color: #222; }<br />
<br />
table thead tr th:first-child, table tfoot tr td:first-child { border-left: none; }<br />
<br />
table thead tr th:last-child, table tfoot tr td:last-child { border-right: none; }<br />
<br />
table tbody tr.even, table tbody tr.alt { background: #f9f9f9; }<br />
<br />
table tbody tr:nth-child(even) { background: #f2f2f2; }<br />
<br />
table tbody tr td { color: #333; padding: 9px 10px; vertical-align: top; border: none; }<br />
<br />
/* Microformats ---------------------- */<br />
ul.vcard { display: inline-block; margin: 0 0 12px 0; border: 1px solid #ddd; padding: 10px; }<br />
ul.vcard li { margin: 0; display: block; }<br />
ul.vcard li.fn { font-weight: bold; font-size: 15px; font-size: 1.5rem; }<br />
<br />
p.vevent span.summary { font-weight: bold; }<br />
p.vevent abbr { cursor: default; text-decoration: none; font-weight: bold; border: none; padding: 0 1px; }</div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/notebook/plasmidsTeam:Cornell/notebook/plasmids2013-09-28T02:16:49Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_wetlab').addClass('active');<br />
});<br />
</script><br />
<div class="row team-bios-container"><br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Plasmid Name</th><th>Part</th><th>Contents/Description</th><th>Origin</th><th>Backbone</th><th>Antibiotic Resistance</th><th>Antifungal Resistance</th></tr><br />
<tr><td>pC13A</td><td>BBa_K523022</td><td>crtEIB</td><td>Kit Plate 1 8P</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13B</td><td>BBa_K539119</td><td>crtY</td><td>Kit Plate 1 12K</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pA13C</td><td>pIVEX2.3d</td><td>T7 promoter/terminator</td><td>Luo Lab</td><td>pIVEX2.3d</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pAK13D</td><td>BBa_I712074</td><td>T7 promoter</td><td>Kit Plate 5 6N</td><td>pSB1AK8</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13E</td><td>BBa_K145001</td><td>T7 polymerase</td><td>Kit Plate 4 2C</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13F</td><td>BBa_J04450</td><td>mRFP in pSB1C3</td><td>T.E. Kit</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAh13G</td><td>pUCATPH</td><td>PtrpC, hph, TtrpC</td><td>Turgeon lab</td><td>pUCATPH</td><td>Ampicillin</td><td>Hygromycin</td></tr><br />
<tr><td>pAg13H</td><td>pNG</td><td>Pgpd (C. h.), nptII</td><td>Turgeon lab</td><td>pNG</td><td>Ampicillin</td><td>Geneticin</td></tr><br />
<tr><td>pC13I</td><td>BBa_K1021000</td><td>crtE</td><td>A</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13J</td><td>invalid</td><td>rbs + crtE</td><td>A</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13K</td><td>BBa_K118003</td><td>crtI</td><td>Kit Plate 3 2B</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13L</td><td>BBa_K118005</td><td>rbs + crtI</td><td>Kit Plate 4 4A</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13M</td><td>BBa_K118002</td><td>crtB</td><td>Kit Plate 3 17N</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13N</td><td>BBa_K118006</td><td>rbs + crtB</td><td>Kit Plate 3 17P</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13O</td><td>BBa_I742155</td><td>rbs + crtY</td><td>Kit Plate 5 17P</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13P</td><td>BBa_K1021007</td><td>PtrpC</td><td>G</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13Q</td><td>BBa_K1021008</td><td>TtrpC</td><td>G</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pCg13R</td><td>invalid</td><td>nptII</td><td>H</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pCg13S</td><td>BBa_K1021001</td><td>nptIIBB</td><td>R</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pAb13T</td><td>pBARGPE1</td><td>PtrpC, bar, TtrpC, PgpdA, lox</td><td>Fungal Genetics Stock Center</td><td>pBARGPE1</td><td>Ampicillin</td><td>Phosphonothricin</td></tr><br />
<tr><td>pCg13U</td><td>invalid</td><td>nptIIBB + TtrpC</td><td>S, Q</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pCh13V</td><td>invalid</td><td>hph</td><td>G, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pCh13W</td><td>BBa_K1021002</td><td>hphBB</td><td>V</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pCp13X</td><td>BBa_K1021003</td><td>bar</td><td>T, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Phosphonothricin</td></tr><br />
<tr><td>pCg13Y</td><td>BBa_K1021015</td><td>PtrpC + nptIIBB</td><td>P, S</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13Z</td><td>BBa_K082003</td><td>GFP(+LVA)</td><td>Kit Plate 3 20N</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13AA</td><td>invalid</td><td>PT7 + crtE</td><td>D, A</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AB</td><td>invalid</td><td>PT7 + crtB</td><td>D, M</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AC</td><td>invalid</td><td>PT7 + crtI</td><td>D, K</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AD</td><td>invalid</td><td>PT7 + crtY</td><td>D, B</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13AE</td><td>BBa_K1021016</td><td>PT7 + crtE</td><td>F, AA</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AF</td><td>BBa_K1021017</td><td>PT7 + crtB</td><td>F, AB</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AG</td><td>BBa_K1021018</td><td>PT7 + crtI</td><td>F, AC</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AH</td><td>BBa_K1021019</td><td>PT7 + crtY</td><td>F, AD</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AI</td><td>BBa_J61046</td><td>lox site</td><td>Kit Plate 3 3L</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pA13AJ</td><td>BBa_J61046</td><td>lox site</td><td>Kit Plate 5 24B</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13AK</td><td>BBa_E1010</td><td>mRFP cds only</td><td>Kit Plate 3 12N</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pK13AL</td><td>BBa_E1010</td><td>mRFP cds only</td><td>Kit Plate 5 18F</td><td>pSB2K3</td><td>Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pA13AM</td><td>BBa_J23100</td><td>strongest Anderson constitutive promoter with mRFP downstream</td><td>Kit Plate 5 18C</td><td>BBa_J61002</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AN</td><td>invalid</td><td>PT7+GFP</td><td>D, Z</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AO</td><td>invalid</td><td>PT7+mRFP</td><td>D, AK/AL</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13AP</td><td>invalid</td><td>PT7+bar</td><td>D, X</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>Phosphonothricin</td></tr><br />
<tr><td>pC13AQ</td><td>BBa_K1021020</td><td>PT7+GFP</td><td>AN, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AR</td><td>BBa_K1021021</td><td>PT7+mRFP</td><td>AO, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AS</td><td>BBa_K1021022</td><td>PT7+bar</td><td>AP, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Phosphonothricin</td></tr><br />
<tr><td>pC13AT</td><td>BBa_K1021023</td><td>PtrpC+GFP</td><td>P, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AU</td><td>BBa_K1021024</td><td>PtrpC+mRFP</td><td>P, AK/AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AV</td><td>BBa_K1021025</td><td>PtrpC+bar</td><td>P, X</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Phosphonothricin</td></tr><br />
<tr><td>pA13AW</td><td>invalid</td><td>Anderson promoter + T7Pol</td><td>AM, E</td><td>BBa_J61002</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pK13AX</td><td>invalid</td><td>Anderson promoter + T7Pol</td><td>AW, AL (backbone)</td><td>pSB2K3</td><td>Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13AY</td><td>unsubmitted</td><td>PtrpC+T7Pol</td><td>P, E</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13AZ</td><td>BBa_K1021026</td><td>Anderson promoter + T7Pol</td><td>AW, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BA</td><td>BBa_K1021004</td><td>afp1</td><td>G-block</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13BB</td><td>invalid</td><td>PT7+nptIIBB</td><td>D, S</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>Geneticin</td></tr><br />
<tr><td>pC13BC</td><td>BBa_K1021032</td><td>PT7+nptIIBB</td><td>BB, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13BD</td><td>unsubmitted</td><td>PtrpC+crtI</td><td>P, K</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pA13BE</td><td>pHNU3pelA</td><td>PpelA, flanked by KpnI/BamHI</td><td>Turgeon lab</td><td>pHPG</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13BF</td><td>BBa_K1021009</td><td>PpelA</td><td>BE, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BG</td><td>BBa_K1021010</td><td>A. nidulans PgpdA</td><td>T, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BH</td><td>invalid</td><td>G. lucidum PgpdA</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BI</td><td>unsubmitted</td><td>G. lucidum PgpdA (BB)</td><td>BH</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BJ</td><td>BBa_K1021011</td><td>100bp left region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BK</td><td>BBa_K1021012</td><td>200bp left region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BL</td><td>BBa_K1021013</td><td>100bp right region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BM</td><td>BBa_K1021014</td><td>200bp right region of homology (Ganoderma gpd gene)</td><td>Ganoderma genome, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BN</td><td>unsubmitted</td><td>lox [cutsite 1] [cutsite 2] lox</td><td>AI</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BO</td><td>abandoned</td><td>short homology regions flanking two unique cutsites</td><td>BJ, BL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BP</td><td>abandoned</td><td>long homology regions flanking two unique cutsites</td><td>BK, BM</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BQ</td><td>BBa_K1021027</td><td>A. nidulans PgpdA + nptIIBB</td><td>BG, S</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13BR</td><td>BBa_K1021028</td><td>A. nidulans PgpdA + hphBB</td><td>BG, W</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13BS</td><td>unsubmitted</td><td>G. lucidum PgpdABB + nptIIBB</td><td>BI, S</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13BT</td><td>unsubmitted</td><td>G. lucidum PgpdABB + hphBB</td><td>BI, W</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pAK13BU</td><td>invalid</td><td>PT7 + hphBB</td><td>D, W</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>Hygromycin</td></tr><br />
<tr><td>pC13BV</td><td>BBa_K1021033</td><td>PT7 + hphBB</td><td>BU, F</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pA13BW</td><td>pBS185CMV-Cre</td><td>pBS185 CMV Cre</td><td>Addgene</td><td>pBS185</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13BX</td><td>BBa_K1021005</td><td>Cre recombinase</td><td>BW</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BY</td><td>unsubmitted</td><td>PpelA + Cre</td><td>BF, BX</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13BZ</td><td>unsubmitted</td><td>PpelA + GFP</td><td>BF, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CA</td><td>unsubmitted</td><td>PpelA + mRFP</td><td>BF, AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CB</td><td>unsubmitted</td><td>PT7 + GFP + PtrpC + nptIIBB</td><td>Y, AQ</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13CC</td><td>BBa_K1021029</td><td>PtrpC + nptIIBB + TtrpC</td><td>Y, Q</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13CD</td><td>BBa_K1021030</td><td>PtrpC + hphBB</td><td>P, W</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13CE</td><td>BBa_K1021034</td><td>PtrpC + hphBB +TtrpC</td><td>CD, Q</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13CF</td><td>unsubmitted</td><td>A. n. PgpdA + GFP</td><td>BG, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CG</td><td>BBa_K1021031</td><td>A. n. PgpdA + mRFP</td><td>BG, AK/AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CH</td><td>unsubmitted</td><td>G. l. PgpdA + GFP</td><td>BH, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CI</td><td>unsubmitted</td><td>G. I. PgpdA + mRFP</td><td>BH, AK/AL</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CJ</td><td>BBa_I742111</td><td>Limonene synthase</td><td>Kit plate 4</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13CK</td><td>unsubmitted</td><td>PT7+Limonene Synthase</td><td>CJ, D</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13CL</td><td>BBa_K1021006</td><td>Cht1_2 in pSB1C3</td><td>IDT Geneblock</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CM</td><td>unsubmitted</td><td>PpelA + Cht1_2</td><td>CL, BF</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pS13CN</td><td>BBa_K112306</td><td>Lambda Holin</td><td>Kit plate 3 2012 BBa_K112306</td><td>BBa_K112950</td><td>Spectinomycin</td><td>N/A</td></tr><br />
<tr><td>pC13CO</td><td>unsubmitted</td><td>Lambda Holin in pSB1C3</td><td>CN</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CP</td><td>unsubmitted</td><td>PpelA+Lambda Holin</td><td>CO</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13CQ</td><td>invalid</td><td>PT7 + rbs + crtE</td><td>D, J</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13CR</td><td>invalid</td><td>PT7 + rbs + crtB</td><td>D, N</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13CS</td><td>invalid</td><td>PT7 + rbs + crtI</td><td>D, L</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pAK13CT</td><td>invalid</td><td>PT7 + rbs + crtY</td><td>D, O</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pC13CU</td><td>unsubmitted</td><td>PT7 + rbs + crtE</td><td>F, CQ</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CV</td><td>unsubmitted</td><td>PT7 + rbs + crtB</td><td>F, CR</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CW</td><td>unsubmitted</td><td>PT7 + rbs + crtI</td><td>F, CS</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13CX</td><td>unsubmitted</td><td>PT7 + rbs + crtY</td><td>F, CT</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pAK13CY</td><td>invalid</td><td>PT7 + rbs + GFP</td><td>D, DB</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pA13CZ</td><td>BBa_B0034</td><td>RBS (strong)</td><td>Kit Plate 5, Well 2M</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pAK13DA</td><td>invalid</td><td>PT7 + rbs + mRFP</td><td>D, DC</td><td>pSB1AK8</td><td>Ampicillin & Kanamycin</td><td>N/A</td></tr><br />
<tr><td>pA13DB</td><td>invalid</td><td>RBS+GFP</td><td>CZ, Z</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pA13DC</td><td>invalid</td><td>RBS+mRFP</td><td>CZ, AK</td><td>pSB1A2</td><td>Ampicillin</td><td>N/A</td></tr><br />
<tr><td>pC13DD</td><td>unsubmitted</td><td>PT7+Limonene Synthase</td><td>F, CK</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DE</td><td>unsubmitted</td><td>lox-PtrpC-nptIIBB-lox</td><td>Y, BN</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13DF</td><td>unsubmitted</td><td>lox-A.PgpdA-nptIIBB-lox</td><td>BN, BQ</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13DG</td><td>unsubmitted</td><td>lox-A.PgpdA-hphBB-lox</td><td>BN, BR</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13DH</td><td>unsubmitted</td><td>lox-PtrpC-hphBB-lox</td><td>BN, CD</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13DI</td><td>unsubmitted</td><td>PtrpC + hphBB + GFP</td><td>CD, Z</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Hygromycin</td></tr><br />
<tr><td>pC13DJ</td><td>unsubmitted</td><td>PT7 + crtB + PT7 + crtI</td><td>AF, AG</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DK</td><td>unsubmitted</td><td>PT7 + crtE + PT7 + crtB + PT7 + crtI</td><td>DJ, AE</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DL</td><td>unsubmitted</td><td>PT7 + crtE + PT7 + crtB + PT7 + crtI + PT7 + nptIIBB</td><td>DK, BC</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
<tr><td>pC13DM</td><td>unsubmitted</td><td>PT7 + rbs + crtB + PT7 + rbs + crtI</td><td>CV, CW</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DN</td><td>unsubmitted</td><td>PT7 + rbs + crtE + PT7 + rbs + crtB + PT7 + rbs + crtI</td><td>DM, CU</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DO</td><td>unsubmitted</td><td>Gan. PgpdA + crtE</td><td>BI, I</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DP</td><td>unsubmitted</td><td>Asp. PgpdA + crtB</td><td>BG, M</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DQ</td><td>unsubmitted</td><td>Asp. PgpdA + crtB + PtrpC + crtI</td><td>DP, BD</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DR</td><td>unsubmitted</td><td>Gan. PgpdA + crtE + Asp. PgpdA + crtB + PtrpC + crtI</td><td>DQ, DO</td><td>pSB1C3</td><td>Chloramphenicol</td><td>N/A</td></tr><br />
<tr><td>pC13DS</td><td>unsubmitted</td><td>Gan. PgpdA + crtE + Asp. PgpdA + crtB + PtrpC + crtI + PtrpC + nptII</td><td>DS, DR</td><td>pSB1C3</td><td>Chloramphenicol</td><td>Geneticin</td></tr><br />
</table><br />
</div><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/templates/headerTeam:Cornell/templates/header2013-09-28T02:14:29Z<p>R.Lizarralde: </p>
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/projectTeam:Cornell/project2013-09-28T02:12:45Z<p>R.Lizarralde: </p>
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<h3 class="centered" style="margin:0px"> We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.</h3><br />
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<h4>Background</h4><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
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<h4>Drylab</h4><br />
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<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab/design_considerations">Design Considerations</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab/modeling">Modeling</a><br />
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<h4>Human Practices</h4><br />
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<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/hprac/ecovative">Collaboration with Ecovative</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/hprac/economics">Economic Analysis</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/hprac/environment">Environmental Impact</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/hprac/future">Future Applications</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/safety">Safety</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/future">Overview</a><br />
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<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/other">Other Biomaterials</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/future/bioremediation">Bioremediation</a><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/templates/backnavTeam:Cornell/templates/backnav2013-09-28T02:12:12Z<p>R.Lizarralde: </p>
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<h6>Background</h6><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2013-09-28T02:11:03Z<p>R.Lizarralde: </p>
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<h2 class="centered">Safety</h2> <br />
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<h3>Specific Safety Concerns</h3><br />
We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a multi-faceted killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.<br />
<h4>Laboratory Safety</h4><br />
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.<br />
<h4>Chassis Organisms</h4><br />
<img style="max-height:1000px;margin:10px" src="https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png"/><br /> <br />
The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities. <br />
<h4>Novel Coding Regions</h4><br />
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.<br />
<img style="max-height:1000px;margin:10px" src="https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png"/> <br />
</div><br />
<br />
<div class="row"><br />
<h3>Safety Protocol</h3><br />
<h4>Wet Lab</h4><br />
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.<br />
<br><br><br />
There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.<br />
<br><br><br />
All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS. <br />
<br><br><br />
We maintain two copies of MSDS sheets for every chemical stock that we maintain in our lab; one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.<br />
<h4>Dry Lab</h4><br />
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects. <br />
</div><br />
<br />
<div class="row"><br />
<h3>Training and Enforcement</h3><br />
<h4>Required Safety Training</h4><br />
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.<br />
<h4>Safety Officers</h4><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/">Institutional Biosafety Committee</a>, to ensure proper equipment usage.<br />
<br><br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.<br />
<br><br><br />
Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
</div><br />
<br />
<div class="row last-ele"><br />
<h3>References</h3><br />
<ol><br />
<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</li><br />
<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx</li><br />
<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</li><br />
</ol> <br />
<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2013-09-28T02:10:16Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_safety').addClass('active');<br />
});<br />
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<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Safety</h2> <br />
</div> <br />
</div><br />
<div class="row"><br />
<h3>Specific Safety Concerns</h3><br />
We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a multi-faceted killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.<br />
<h4>Laboratory Safety</h4><br />
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.<br />
<h4>Chassis Organisms</h4><img style="max-height:1000px" src="https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png"/><br /> <br />
The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities. <br />
<h4>Novel Coding Regions</h4><br />
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.<br />
<img style="max-height:1000px" src="https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png"/> <br />
</div><br />
<br />
<div class="row"><br />
<h3>Safety Protocol</h3><br />
<h4>Wet Lab</h4><br />
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.<br />
<br><br><br />
There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.<br />
<br><br><br />
All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS. <br />
<br><br><br />
We maintain two copies of MSDS sheets for every chemical stock that we maintain in our lab; one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.<br />
<h4>Dry Lab</h4><br />
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects. <br />
</div><br />
<br />
<div class="row"><br />
<h3>Training and Enforcement</h3><br />
<h4>Required Safety Training</h4><br />
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.<br />
<h4>Safety Officers</h4><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/">Institutional Biosafety Committee</a>, to ensure proper equipment usage.<br />
<br><br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.<br />
<br><br><br />
Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
</div><br />
<br />
<div class="row last-ele"><br />
<h3>References</h3><br />
<ol><br />
<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</li><br />
<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx</li><br />
<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</li><br />
</ol> <br />
<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2013-09-28T02:09:40Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_safety').addClass('active');<br />
});<br />
</script><br />
<div class="row"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/hpracnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Safety</h2> <br />
</div> <br />
</div><br />
<div class="row"><br />
<h3>Specific Safety Concerns</h3><br />
We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a multi-faceted killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.<br />
<h4>Laboratory Safety</h4><br />
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.<br />
<h4>Chassis Organisms</h4><br />
<img style="max-height:1000px" src="https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png"/> <br />
The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities. <br />
<h4>Novel Coding Regions</h4><br />
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.<br />
<img style="max-height:1000px" src="https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png"/> <br />
</div><br />
<br />
<div class="row"><br />
<h3>Safety Protocol</h3><br />
<h4>Wet Lab</h4><br />
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.<br />
<br><br><br />
There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.<br />
<br><br><br />
All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS. <br />
<br><br><br />
We maintain two copies of MSDS sheets for every chemical stock that we maintain in our lab; one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.<br />
<h4>Dry Lab</h4><br />
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects. <br />
</div><br />
<br />
<div class="row"><br />
<h3>Training and Enforcement</h3><br />
<h4>Required Safety Training</h4><br />
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.<br />
<h4>Safety Officers</h4><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/">Institutional Biosafety Committee</a>, to ensure proper equipment usage.<br />
<br><br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.<br />
<br><br><br />
Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
</div><br />
<br />
<div class="row last-ele"><br />
<h3>References</h3><br />
<ol><br />
<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</li><br />
<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx</li><br />
<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</li><br />
</ol> <br />
<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2013-09-28T02:08:59Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_safety').addClass('active');<br />
});<br />
</script><br />
<div class="row"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/hpracnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Safety</h2> <br />
</div> <br />
</div><br />
<div class="row"><br />
<h3>Specific Safety Concerns</h3><br />
We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a multi-faceted killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.<br />
<h4>Laboratory Safety</h4><br />
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.<br />
<h4>Chassis Organisms</h4><br />
<img style="max-height:1000px" src="https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png"/> <br />
<br><br><br />
The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities. <br />
<h4>Novel Coding Regions</h4><br />
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.<br />
<br><br><br />
<img style="max-height:1000px" src="https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png"/> <br />
</div><br />
<br />
<div class="row"><br />
<h3>Safety Protocol</h3><br />
<h4>Wet Lab</h4><br />
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.<br />
<br><br><br />
There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.<br />
<br><br><br />
All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS. <br />
<br><br><br />
We maintain two copies of MSDS sheets for every chemical stock that we maintain in our lab; one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.<br />
<h4>Dry Lab</h4><br />
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects. <br />
</div><br />
<br />
<div class="row"><br />
<h3>Training and Enforcement</h3><br />
<h4>Required Safety Training</h4><br />
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.<br />
<h4>Safety Officers</h4><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/">Institutional Biosafety Committee</a>, to ensure proper equipment usage.<br />
<br><br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.<br />
<br><br><br />
Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
</div><br />
<br />
<div class="row last-ele"><br />
<h3>References</h3><br />
<ol><br />
<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</li><br />
<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx</li><br />
<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</li><br />
</ol> <br />
<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2013-09-28T02:06:14Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_safety').addClass('active');<br />
});<br />
</script><br />
<div class="row"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/hpracnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Safety</h2> <br />
</div> <br />
</div><br />
<div class="row"><br />
<h3>Specific Safety Concerns</h3><br />
We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a multi-faceted killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.<br />
<h4>Laboratory Safety</h4><br />
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.<br />
<h4>Chassis Organisms</h4><br />
<img src="https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png"/> <br />
<br><br><br />
The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities. <br />
<h4>Novel Coding Regions</h4><br />
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.<br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png"/> <br />
</div><br />
<br />
<div class="row"><br />
<h3>Safety Protocol</h3><br />
<h4>Wet Lab</h4><br />
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.<br />
<br><br><br />
There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.<br />
<br><br><br />
All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS. <br />
<br><br><br />
We maintain two copies of MSDS sheets for every chemical stock that we maintain in our lab; one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.<br />
<h4>Dry Lab</h4><br />
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects. <br />
</div><br />
<br />
<div class="row"><br />
<h3>Training and Enforcement</h3><br />
<h4>Required Safety Training</h4><br />
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.<br />
<h4>Safety Officers</h4><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/">Institutional Biosafety Committee</a>, to ensure proper equipment usage.<br />
<br><br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.<br />
<br><br><br />
Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
</div><br />
<br />
<div class="row last-ele"><br />
<h3>References</h3><br />
<ol><br />
<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</li><br />
<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx</li><br />
<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</li><br />
</ol> <br />
<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/safetyTeam:Cornell/project/safety2013-09-28T02:04:15Z<p>R.Lizarralde: </p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project_safety').addClass('active');<br />
});<br />
</script><br />
<div class="row"><br />
<div class="two columns"></html>{{:Team:Cornell/templates/hpracnav}}<html></div> <br />
<div class="ten columns team-bios-container"><br />
<div class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Safety</h2> <br />
</div> <br />
</div><br />
<div class="row"><br />
<h3>Specific Safety Concerns</h3><br />
We must be concerned with the safety implications that our project could subsequently have on the natural environment, as we are modifying the genomes of organisms to produce novel functions. We have designed and are implementing a number of <a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">biological safety mechanisms</a> to prevent the spread of potentially harmful genetic material to the environment. This includes a recombination system to remove and degrade engineered portions of fungal genomes before using the fungus for packaging production, as well as a multi-faceted killswitch to induce our organism to halt its own growth upon the addition of a very specific chemical cue. We have corresponded with our corporate collaborators to determine the sorts of precautions that may be necessary for commercial deployment and are working to incorporate them.<br />
<h4>Laboratory Safety</h4><br />
Our project involves regular use of ethidium bromide, a DNA-intercalating agent known to cause cancer, as well as the use of powerful UV light, for visualization of gel electrophoresis. We must prepare culture media with antibiotics, which could be harmful to humans in large doses. We also work with ethanol lamps to maintain a sterile environment, which do involve having an open flame on the benchtop.<br />
<h4>Chassis Organisms</h4><br />
<img src=”https://static.igem.org/mediawiki/2013/9/9c/Chassis_safety.png”/> <br />
<br><br><br />
The wet lab subteam regularly works with a non-pathogenic strain of <i>E. coli</i>; as part of our project this year, we are also working with two fungal species, <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>, as well as <i>Agrobacterium tumefaciens</i> and <i>Aspergillus niger</i>, to a limited degree. These are all Biosafety Level 1 organisms and are therefore safe to work with within our existing lab space; however, these all require special precautions. Fungi, for instance, can release spores that can be irritating to eyes, skin and lungs, and damaging if inhaled or ingested in significant quantities. <br />
<h4>Novel Coding Regions</h4><br />
None of our novel coding regions pose a threat to researcher safety; however, genes such as those for chitinase and antifungal selection markers could be harmful if released into the environment. We must therefore use care to ensure that our engineered organisms do not escape the confines of our lab space.<br />
<br><br><br />
<img src=”https://static.igem.org/mediawiki/2013/5/50/Cds_safety.png”/> <br />
</div><br />
<br />
<div class="row"><br />
<h3>Safety Protocol</h3><br />
<h4>Wet Lab</h4><br />
In the lab all members wear nitrile gloves, closed-toed shoes, and use eye protection when working with volatile chemicals or using UV light. Members typically work in groups of 2-3 so as to be aware of each other’s safety and the work at hand. If using a new piece of equipment, a team member must be supervised by either the faculty lab manager or, in her absence, an experienced team member.<br />
<br><br><br />
There are taped-off areas for work with carcinogenic substances (particularly ethidium bromide) as well as fungal specimens. When working with fungi that could potentially release harmful spores, team members must work in a designated area and wear masks and eye protection. The delineated and surrounding areas are cleaned with ethanol and/or bleach prior to and immediately following each use, and if these substances must come into contact with a non-designated area, it is cleaned thoroughly to ensure that no residue remains.<br />
<br><br><br />
All disposables that come into contact with biological materials, including but not limited to: pipet tips, syringes, filters, membranes, Petri dishes, microcentrifuge tubes, culture tubes, and gloves, are disposed of in the biohazard waste. There are also specialized waste containers for glass and sharps accessible in the lab space. Though we are not working with any highly toxic substances this year, we have protocols in place for using and collecting waste from hazardous chemical and/or biological substances in a manner approved by Cornell EHS. <br />
<br><br><br />
We maintain two copies of MSDS sheets for every chemical stock that we maintain in our lab; one for our own reference and the other for the lab manager and users of the lab space who are not team members. There is a chemicals hood available for use with any chemicals that may produce harmful fumes, i.e. acids and organics. The lab is also equipped with flame-retardant benches, spill kits, safety showers, eyewashes, fire extinguishers, and clearly marked emergency exits.<br />
<h4>Dry Lab</h4><br />
We use the Emerson Machine Shop for fabrication; each of the drylab subteam members has attended the prescribed training session for use of the shop and has learned to use each of the tools safely. Each member of the drylab subteam was trained in the safe usage of the milling machine and the metal lathe. <br />
<br><br><br />
All machine shop work is conducted under the supervision of the Emerson machine shop staff. Safety goggles were worn at all times. Masks and gloves are worn as appropriate. Closed toe shoes and long pants were also worn when working in the machine shop. While working in the machine shop we maintained a clean work environment so we could maintain visibility at all times. When lifting heavy objects, proper lifting technique was used, and an appropriate number of individuals were used for lifting said objects. <br />
</div><br />
<br />
<div class="row"><br />
<h3>Training and Enforcement</h3><br />
<h4>Required Safety Training</h4><br />
All team members are required to take the EHS Chemical Safety and Laboratory Safety Training, as well as an orientation session with the building coordinator. In addition, before gaining access to the lab, all team members go through an orientation with our lab's manager detailing proper equipment usage and safety within our lab space. Before any independent lab work begins, wet lab members spend the first week of every summer project in a laboratory Boot Camp hosted by experienced team members and, if needed, graduate advisers. These training sessions ensure proper laboratory technique, protocol, attire, and safety. Dry lab members are required to undergo machine shop training in order to gain access to the Emerson machine shop, to similarly ensure proper equipment usage.<br />
<h4>Safety Officers</h4><br />
The safety officers were chosen to be team members who could directly supervise the activities of the other team members. One team member each was chosen for the wet and dry lab subteams to ensure that all team members are working safely, whether with bacterial cultures or power tools. These team members also act as liaisons to the wet lab and machine shop managers and, when necessary, the <a href="http://www.ibc.cornell.edu/">Institutional Biosafety Committee</a>, to ensure proper equipment usage.<br />
<br><br><br />
These team members are responsible for discussing the proposed workplan for the project with the wet lab and machine shop managers before starting work to ensure that it is safe to continue. In the case of the wet lab in particular, this involves going through a detailed list of protocols, including all organisms, chemicals, and genetic constructs being worked with, to ensure conformity with the <a href="http://sp.ehs.cornell.edu/lab-research-safety/bios/biological-safety-manuals/Pages/default.aspx">Environmental Health & Safety guidelines</a>. They must go through the same safety training as all other team members, but are required to redo the training each time we recruit new members in order to keep up-to-date with safety considerations. In addition, they maintain contact with the supervisors of the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns that have arisen and ensure that equipment continues to be used properly.<br />
<br><br><br />
Team members who violate safety rules are required to work under supervision of the safety officers for the remainder of the week, or until the safety officer believes the member is capable of perform the task unsupervised. For multiple infractions or complete disregard to safety protocols, a member may be restricted from laboratory work until he/she undergoes EHS chemical safety online training again, and demonstrates proper performance to a team leader of failed technique(s) in a controlled setting.<br />
</div><br />
<br />
<div class="row last-ele"><br />
<h3>References</h3><br />
<ol><br />
<li>Lawrence Berkeley National Lab (2011) <i>Appendix B Pathogen and Toxin Lists</i> Retrieved from: http://www.lbl.gov/ehs/biosafety/manual/html/AppxB.shtml</li><br />
<li>Product Sheet Ganoderma lucidum ATCC® 32471™ (2013) Retrieved from: http://www.atcc.org/~/ps/32471.ashx</li><br />
<li>University of Pittsburgh (2013) <i>Risk Group and Biosafety Chart</i>&nbsp;Retrieved from: http://www.ibc.pitt.edu/RiskGroups/RiskBiosafetyChart2007.htm</li><br />
</ol> <br />
<br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/projectTeam:Cornell/project2013-09-28T02:02:56Z<p>R.Lizarralde: moved protoplasting up</p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project').addClass('active');<br />
});<br />
</script><br />
<div class="row main-image"><br />
<div class="twelve columns"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/CornellMicroryza940x329.png" /><br />
<a class="button radius gallery-button" href="https://2013.igem.org/Team:Cornell/project/gallery">More Photos</a><br />
</div><br />
</div><br />
<div class="row" style="margin-top:20px;"><br />
<div class="twelve columns"><br />
<div class="row"><br />
<div class="eleven columns centered panel"><br />
<h3 class="centered" style="margin:0px"> We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.</h3><br />
</div><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="four columns"><br />
<h4>Background</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/fungal_toolkit">Fungal Toolkit</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/fungal_genetic_engineering">Fungal Genetic Engineering</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/ecovative">Ecovative</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/impact">Environmental Impact</a><br />
</li><br />
</ul><br />
</div><br />
<div class="four columns"><br />
<h4>Wetlab</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/protoplasting">Protoplasting</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit">Fungal Toolkit</a><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/regulatory_elements">Regulatory Elements</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/selectable_markers">Selectable Markers</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/characterization">Characterization</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/homologous_constructs">Homologous Constructs</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">Biosafety</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/carotenoids">Carotenoids</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/antifungals">Antifungals</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
</ul><br />
</div><br />
<div class="four columns"><br />
<h4>Drylab</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/design_considerations">Design Considerations</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/components">Components</a><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/components/fabrication">Fabrication</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/components/electronics">Electronics</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/modeling">Modeling</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="four columns"><br />
<h4>Human Practices</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/ecovative">Collaboration with Ecovative</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/economics">Economic Analysis</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/environment">Environmental Impact</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/ethics">Bioethics</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/future">Future Applications</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
<div class="four columns"><br />
<h4>Future Applications</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/other">Other Biomaterials</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/bioremediation">Bioremediation</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/pharmaceuticals">Pharmeceuticals</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/projectTeam:Cornell/project2013-09-28T02:01:35Z<p>R.Lizarralde: fixed centered titles</p>
<hr />
<div>{{:Team:Cornell/templates/header}}<br />
<html class="no-js" lang="en"><br />
<script type="text/javascript"><br />
$(window).load(function() {<br />
$('li.pg-project').addClass('active');<br />
});<br />
</script><br />
<div class="row main-image"><br />
<div class="twelve columns"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/CornellMicroryza940x329.png" /><br />
<a class="button radius gallery-button" href="https://2013.igem.org/Team:Cornell/project/gallery">More Photos</a><br />
</div><br />
</div><br />
<div class="row" style="margin-top:20px;"><br />
<div class="twelve columns"><br />
<div class="row"><br />
<div class="eleven columns centered panel"><br />
<h3 class="centered" style="margin:0px"> We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.</h3><br />
</div><br />
</div><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="four columns"><br />
<h4>Background</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/fungal_toolkit">Fungal Toolkit</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/fungal_genetic_engineering">Fungal Genetic Engineering</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/ecovative">Ecovative</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/background/impact">Environmental Impact</a><br />
</li><br />
</ul><br />
</div><br />
<div class="four columns"><br />
<h4>Wetlab</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit">Fungal Toolkit</a><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/protoplasting">Protoplasting</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/regulatory_elements">Regulatory Elements</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/selectable_markers">Selectable Markers</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/characterization">Characterization</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/homologous_constructs">Homologous Constructs</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">Biosafety</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/carotenoids">Carotenoids</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/antifungals">Antifungals</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/biobricks">BioBricks</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
</li><br />
</ul><br />
</div><br />
<div class="four columns"><br />
<h4>Drylab</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/design_considerations">Design Considerations</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/components">Components</a><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/components/fabrication">Fabrication</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/components/electronics">Electronics</a><br />
</li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/modeling">Modeling</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="four columns"><br />
<h4>Human Practices</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/ecovative">Collaboration with Ecovative</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/economics">Economic Analysis</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/environment">Environmental Impact</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/ethics">Bioethics</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/hprac/future">Future Applications</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
<div class="four columns"><br />
<h4>Future Applications</h4><br />
<ul><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future">Overview</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/other">Other Biomaterials</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/bioremediation">Bioremediation</a><br />
</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/future/pharmaceuticals">Pharmeceuticals</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/stylesheets/appTeam:Cornell/stylesheets/app2013-09-28T01:56:46Z<p>R.Lizarralde: </p>
<hr />
<div>/* --------------------------------------------------<br />
Table of Contents<br />
-----------------------------------------------------<br />
::iGEM Reset<br />
::Fonts and Icons<br />
::Lightbox<br />
::Shared Styles<br />
::Header & Footer<br />
::Homepage<br />
::Team page<br />
::Tour Template<br />
::Notebook page<br />
::Gallery page<br />
::Functional Requirements: Hover-overs<br />
::Device Hover Overs<br />
*/<br />
<br />
/* -----------------------------------------<br />
iGEM Reset<br />
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background-color: transparent;<br />
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margin: 0;<br />
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width: 100%;<br />
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}<br />
#content {<br />
background-color: transparent;<br />
border: none;<br />
padding: 0;<br />
margin: 0;<br />
width: 100%;<br />
overflow: visible;<br />
height: 100%;<br />
}<br />
#bodyContent {<br />
border: none;<br />
padding: 0;<br />
margin: 0;<br />
width: 100%;<br />
height: 100%;<br />
}<br />
#top-section {<br />
height: 15px;<br />
margin: 0px;<br />
margin-left: auto;<br />
margin-right: auto;<br />
margin-bottom: 0 !important;<br />
padding: 0;<br />
border: none;<br />
font-size: 10px;<br />
}<br />
#p-logo {<br />
height: 1px;<br />
overflow: hidden;<br />
display: none;<br />
}<br />
#search-controls {<br />
overflow: hidden;<br />
display: block;<br />
background: none;<br />
position: absolute;<br />
top: 100px;<br />
right: 40px;<br />
}<br />
#menubar.left-menu {<br />
/*margin-top: -80px; uncomment this when page goes live */<br />
background: none;<br />
height: 20px;<br />
left: 20px;<br />
float: left;<br />
}<br />
.left-menu ul {<br />
visibility: hidden;<br />
}<br />
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visibility: visible;<br />
}<br />
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color: #666;<br />
}<br />
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color: #aaaaff !important;<br />
text-decoration: underline;<br />
}<br />
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display: block;<br />
width: 300px;<br />
float: left;<br />
margin-top: 2px;<br />
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.right-menu ul {<br />
width: 300px;<br />
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width: 100%;<br />
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display: none;<br />
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border: none;<br />
width: 965px;<br />
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color: #666;<br />
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margin: 0;<br />
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}<br />
#search-controls {<br />
display: none;<br />
}<br />
h3#siteSub {<br />
display: none;<br />
}<br />
#contentSub {<br />
display: none;<br />
}<br />
p:first-child {<br />
display: none;<br />
}<br />
#catlinks {<br />
display: none;<br />
}<br />
#footer-box {<br />
width: 977px;<br />
margin-bottom: 20px;<br />
}<br />
<br />
/* -----------------------------------------<br />
Fonts and Icons<br />
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<br />
@font-face {<br />
font-family: 'Bitter';<br />
font-style: normal;<br />
font-weight: 400;<br />
src: local('Bitter-Regular'), url(http://themes.googleusercontent.com/static/fonts/bitter/v4/2PcBT6-VmYhQCus-O11S5-vvDin1pK8aKteLpeZ5c0A.woff) format('woff');<br />
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font-family: 'Bitter';<br />
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src: local('Bitter-Bold'), url(http://themes.googleusercontent.com/static/fonts/bitter/v4/evC1haE-MsorTl_A7_uSGbO3LdcAZYWl9Si6vvxL-qU.woff) format('woff');<br />
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/*<br />
@font-face {<br />
font-family: 'Foro-LightRegular';<br />
src: url('https://dl.dropbox.com/u/3824307/iGEM/fonts/forolig-webfont.eot');<br />
src: url('https://dl.dropbox.com/u/3824307/iGEM/fonts/forolig-webfont.eot?#iefix') format('embedded-opentype'), url('https://dl.dropbox.com/u/3824307/iGEM/fonts/forolig-webfont.woff') format('woff'), url('https://dl.dropbox.com/u/3824307/iGEM/fonts/forolig-webfont.ttf') format('truetype'), url('https://dl.dropbox.com/u/3824307/iGEM/fonts/forolig-webfont.svg#Foro-LightRegular') format('svg');<br />
font-weight: normal;<br />
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@font-face {<br />
font-family: 'Droid Sans';<br />
font-style: normal;<br />
font-weight: 400;<br />
src: local('Droid Sans'), local('DroidSans'), url(http://themes.googleusercontent.com/static/fonts/droidsans/v3/s-BiyweUPV0v-yRb-cjciBsxEYwM7FgeyaSgU71cLG0.woff) format('woff');<br />
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@font-face {<br />
font-family: 'Droid Sans';<br />
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src: local('Droid Sans Bold'), local('DroidSans-Bold'), url(http://themes.googleusercontent.com/static/fonts/droidsans/v3/EFpQQyG9GqCrobXxL-KRMQFhaRv2pGgT5Kf0An0s4MM.woff) format('woff');<br />
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/* -----------------------------------------<br />
Lightbox<br />
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/* line 6, ../sass/lightbox.sass */<br />
#lightboxOverlay {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
z-index: 9999;<br />
background-color: black;<br />
filter: progid:DXImageTransform.Microsoft.Alpha(Opacity=85);<br />
opacity: 0.85;<br />
display: none;<br />
}<br />
<br />
/* line 15, ../sass/lightbox.sass */<br />
#lightbox {<br />
position: absolute;<br />
left: 0;<br />
width: 100%;<br />
z-index: 10000;<br />
text-align: center;<br />
line-height: 0;<br />
font-family: "lucida grande", tahoma, verdana, arial, sans-serif;<br />
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}<br />
/* line 24, ../sass/lightbox.sass */<br />
#lightbox img {<br />
width: auto;<br />
height: auto;<br />
}<br />
/* line 27, ../sass/lightbox.sass */<br />
#lightbox a img {<br />
border: none;<br />
}<br />
<br />
/* line 30, ../sass/lightbox.sass */<br />
.lb-outerContainer {<br />
position: relative;<br />
background-color: white;<br />
*zoom: 1;<br />
width: 250px;<br />
height: 250px;<br />
margin: 0 auto;<br />
-webkit-border-radius: 4px;<br />
-moz-border-radius: 4px;<br />
-ms-border-radius: 4px;<br />
-o-border-radius: 4px;<br />
border-radius: 4px;<br />
}<br />
/* line 38, ../../../../.rvm/gems/ruby-1.9.2-p290/gems/compass-0.12.1/frameworks/compass/stylesheets/compass/utilities/general/_clearfix.scss */<br />
.lb-outerContainer:after {<br />
content: "";<br />
display: table;<br />
clear: both;<br />
}<br />
<br />
/* line 39, ../sass/lightbox.sass */<br />
.lb-container {<br />
padding: 10px;<br />
}<br />
<br />
/* line 42, ../sass/lightbox.sass */<br />
.lb-loader {<br />
position: absolute;<br />
top: 40%;<br />
left: 0%;<br />
height: 25%;<br />
width: 100%;<br />
text-align: center;<br />
line-height: 0;<br />
}<br />
<br />
/* line 51, ../sass/lightbox.sass */<br />
.lb-nav {<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
height: 100%;<br />
width: 100%;<br />
z-index: 10;<br />
}<br />
<br />
/* line 59, ../sass/lightbox.sass */<br />
.lb-container > .nav {<br />
left: 0;<br />
}<br />
<br />
/* line 62, ../sass/lightbox.sass */<br />
.lb-nav a {<br />
outline: none;<br />
}<br />
<br />
/* line 65, ../sass/lightbox.sass */<br />
.lb-prev, .lb-next {<br />
width: 49%;<br />
height: 100%;<br />
background-image: url("data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7");<br />
/* Trick IE into showing hover */<br />
display: block;<br />
}<br />
<br />
/* line 72, ../sass/lightbox.sass */<br />
.lb-prev {<br />
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}<br />
<br />
/* line 76, ../sass/lightbox.sass */<br />
.lb-next {<br />
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<br />
/* line 81, ../sass/lightbox.sass */<br />
.lb-prev:hover {<br />
background: url(https://static.igem.org/mediawiki/2013/3/30/Cornell-lightbox-Prev.png) left 48% no-repeat;<br />
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<br />
/* line 85, ../sass/lightbox.sass */<br />
.lb-next:hover {<br />
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<br />
/* line 88, ../sass/lightbox.sass */<br />
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padding-top: 5px;<br />
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/* line 38, ../../../../.rvm/gems/ruby-1.9.2-p290/gems/compass-0.12.1/frameworks/compass/stylesheets/compass/utilities/general/_clearfix.scss */<br />
.lb-dataContainer:after {<br />
content: "";<br />
display: table;<br />
clear: both;<br />
}<br />
<br />
/* line 95, ../sass/lightbox.sass */<br />
.lb-data {<br />
padding: 0 10px;<br />
color: #bbbbbb;<br />
}<br />
/* line 98, ../sass/lightbox.sass */<br />
.lb-data .lb-details {<br />
width: 85%;<br />
float: left;<br />
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line-height: 1.1em;<br />
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/* line 103, ../sass/lightbox.sass */<br />
.lb-data .lb-caption {<br />
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font-weight: bold;<br />
line-height: 1em;<br />
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/* line 107, ../sass/lightbox.sass */<br />
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padding-bottom: 1em;<br />
font-size: 11px;<br />
}<br />
/* line 112, ../sass/lightbox.sass */<br />
.lb-data .lb-close {<br />
width: 35px;<br />
float: right;<br />
padding-bottom: 0.7em;<br />
outline: none;<br />
}<br />
/* line 117, ../sass/lightbox.sass */<br />
.lb-data .lb-close:hover {<br />
cursor: pointer;<br />
}<br />
<br />
/* -----------------------------------------<br />
Shared Styles<br />
----------------------------------------- */<br />
<br />
.nohilite {<br />
-webkit-touch-callout: none;<br />
-webkit-user-select: none;<br />
-khtml-user-select: none;<br />
-moz-user-select: none;<br />
-ms-user-select: none;<br />
user-select: none;<br />
}<br />
<br />
<br />
body {<br />
background: #f2f2f2;<br />
font-family: 'Droid Sans', Helvetica, sans-serif;<br />
font-size: 15px;<br />
line-height: 23px;<br />
}<br />
<br />
h1, h2, h3, h4, h5, h6 {<br />
font-family: 'Bitter', serif;<br />
font-weight: normal;<br />
color: #000;<br />
border-bottom: none;<br />
margin-bottom: none;<br />
}<br />
<br />
h1.centered, h2.centered, h3.centered, h4.centered, h5.centered, h6.centered {<br />
text-align: center;<br />
}<br />
<br />
div.clearfix {<br />
content: ".";<br />
display: block;<br />
clear: both;<br />
visibility: hidden;<br />
line-height: 0;<br />
height: 0;<br />
}<br />
<br />
div.twelve.columns.no-pad {<br />
padding: 0px;<br />
}<br />
<br />
.off-canvas {<br />
width: 100%;<br />
}<br />
<br />
a, a:visited {<br />
color: #b31b1b;<br />
text-decoration: none;<br />
}<br />
<br />
a:hover {<br />
color: #871414;<br />
text-decoration: none;<br />
}<br />
<br />
.button {<br />
background: #b31b1b;<br />
border: 1px solid #691010;<br />
color: #fff;<br />
}<br />
<br />
a.button, a.button:hover {<br />
color: #fff;<br />
}<br />
<br />
.button:hover {<br />
background-color: #871414;<br />
}<br />
<br />
.button:focus {<br />
-webkit-box-shadow: 0 0 4px #b31b1b, 0 1px 0 rgba(255,255,255,0.5) inset;<br />
-moz-box-shadow: 0 0 4px #b31b1b, 0 1px 0 rgba(255,255,255,0.5) inset;<br />
box-shadow: 0 0 4px #b31b1b, 0 1px 0 rgba(255,255,255,0.5) inset;<br />
}<br />
<br />
.nav-bar {<br />
background: #b31b1b;<br />
}<br />
<br />
.nav-bar > li {<br />
border: 1px solid #871414;<br />
}<br />
<br />
.nav-bar > li:last-child {<br />
border-right: solid 1px #871414;<br />
}<br />
<br />
.nav-bar > li.active {<br />
background: #4c0b0b;<br />
border-color: #4c0b0b;<br />
}<br />
<br />
.nav-bar > li.inactive {<br />
background: #b31b1b;<br />
border-color: #b31b1b;<br />
font-weight: normal;<br />
}<br />
<br />
.nav-bar > li.active:hover {<br />
background: #4c0b0b;<br />
}<br />
<br />
.nav-bar > li:hover {<br />
background: #871414;<br />
}<br />
<br />
.nav-bar a {<br />
color: #f2f2f2;<br />
}<br />
<br />
ul.side-nav li.divider {<br />
border-top: 1px solid #ddd;<br />
}<br />
<br />
dl.tabs.pill dd a {<br />
background: #ddd;<br />
color: #000;<br />
}<br />
<br />
dl.tabs.pill dd.active a {<br />
background-color: #b31b1b;<br />
color: #e6e6e6;<br />
}<br />
<br />
iframe.slides {<br />
width: 577px;<br />
height: 476px;<br />
margin: 5px auto;<br />
}<br />
/* -----------------------------------------<br />
Header & Footer<br />
----------------------------------------- */<br />
<br />
div.row.header {<br />
background: #b31b1b;<br />
margin: 20px auto 40px;<br />
}<br />
<br />
div#header-wrapper {<br />
width: 940px;<br />
margin: 0px auto;<br />
}<br />
<br />
div#logo-wrapper {<br />
position: absolute;<br />
top: -20px;<br />
left: 0px;<br />
background: green;<br />
border: 10px solid #b31b1b;<br />
height: 80px;<br />
}<br />
<br />
div#logo-wrapper img {<br />
height: 60px;<br />
}<br />
<br />
div#igem-attr {<br />
position: absolute;<br />
top: -12px;<br />
left: 147px;<br />
height: 60px;<br />
transition: transform 0.5s;<br />
-moz-transition: -moz-transform 0.5s; /* Firefox 4 */<br />
-webkit-transition: -webkit-transform 0.5s; /* Safari and Chrome */<br />
-o-transition: -o-transform 0.5s; /* Opera */<br />
}<br />
<br />
div#igem-attr:hover {<br />
transform: rotate(-15deg);<br />
-ms-transform: rotate(-15deg); /* IE 9 */<br />
-webkit-transform: rotate(-15deg); /* Safari and Chrome */<br />
-o-transform: rotate(-15deg); /* Opera */<br />
-moz-transform: rotate(-15deg); /* Firefox */<br />
}<br />
<br />
div#igem-attr img {<br />
height: 60px;<br />
}<br />
<br />
div#header-wrapper .nav-bar {<br />
float: right;<br />
margin: 0px;<br />
}<br />
<br />
div.main-image > div {<br />
padding: 0px;<br />
}<br />
<br />
a.gallery-button {<br />
position: absolute;<br />
right: 20px;<br />
bottom: 10px;<br />
}<br />
<br />
div.missing {<br />
margin: auto;<br />
position: relative;<br />
top: 50%;<br />
width: 193px;<br />
}<br />
div.missing a {<br />
height: 63px;<br />
position: relative;<br />
top: -31px;<br />
}<br />
div.missing img {<br />
border-width: 0px;<br />
}<br />
div#unityPlayer {<br />
cursor: default;<br />
height: 600px;<br />
width: 960px;<br />
}<br />
<br />
/* -----------------------------------------<br />
Home Page<br />
----------------------------------------- */<br />
<br />
div#homepage-slideshow {<br />
margin-bottom: 30px;<br />
}<br />
<br />
div.homepage-section {<br />
padding: 10px;<br />
}<br />
<br />
div.homepage-force {<br />
height: 174px;<br />
}<br />
<br />
div.controls {<br />
position: absolute;<br />
bottom: 0px;<br />
}<br />
<br />
div.controls a.button {<br />
float: right;<br />
}<br />
<br />
div.homepage-section.tl {<br />
border-bottom: 1px solid #ddd;<br />
border-right: 1px solid #ddd;<br />
padding-top: 0px;<br />
padding-left: 0px;<br />
}<br />
<br />
div.homepage-section.tr {<br />
border-bottom: 1px solid #ddd;<br />
padding-top: 0px;<br />
padding-right: 0px;<br />
}<br />
<br />
div.homepage-section.bl {<br />
border-right: 1px solid #ddd;<br />
padding-bottom: 0px;<br />
padding-left: 0px;<br />
}<br />
<br />
div.homepage-section.br {<br />
padding-bottom: 0px;<br />
padding-right: 0px;<br />
}<br />
<br />
div.homepage-thumb {<br />
margin-bottom: -6px;<br />
}<br />
<br />
div.sponsors-wrapper {<br />
margin-top: 20px;<br />
}<br />
<br />
div.sponsors-wrapper ul {<br />
padding: 5px 0px;<br />
}<br />
<br />
div.sponsors-wrapper ul li {<br />
padding: 5px 10px;<br />
display: inline-block;<br />
list-style: none;<br />
}<br />
<br />
div.sponsors-wrapper ul li img {<br />
height: 105px;<br />
display: block;<br />
margin: 5px auto;<br />
}<br />
<br />
/* -----------------------------------------<br />
Team Page<br />
----------------------------------------- */<br />
<br />
div.team-bios-container div.row {<br />
padding: 0px 0px 10px;<br />
border-bottom: 1px solid #ddd;<br />
}<br />
<br />
div.team-bios-container div.last-ele {<br />
border-bottom: none;<br />
}<br />
<br />
div.team-bios-container img {<br />
max-height: 166px;<br />
margin: 29px 0px;<br />
}<br />
<br />
div.team-bios-container img.inline {<br />
max-height: none;<br />
}<br />
<br />
div#grd-members, div#fac-members {<br />
margin-top: 80px;<br />
}<br />
<br />
div.bio-pic-container {<br />
position: relative;<br />
margin: 29px 0px;<br />
height: 166px;<br />
}<br />
<br />
div.bio-pic-container img {<br />
position: absolute;<br />
margin: 0px;<br />
top: 0px;<br />
z-index: 2;<br />
}<br />
<br />
div.bio-pic-container img.bio-pic-bw {<br />
z-index: 5;<br />
}<br />
/* -----------------------------------------<br />
Tour Template<br />
----------------------------------------- */<br />
div.tour {<br />
position: fixed; <br />
bottom: 0px;<br />
margin: 20px;<br />
z-index: 10;<br />
}<br />
<br />
div.tour a {<br />
font-size:14px; <br />
}<br />
/* -----------------------------------------<br />
Notebook Page<br />
----------------------------------------- */<br />
div.author-hover {<br />
z-index: 5;<br />
border-radius: 300px;<br />
cursor: pointer;<br />
display: inline-block;<br />
color: #5555AA;<br />
}<br />
<br />
div.author-hover-pic {<br />
position: absolute;<br />
z-index: 0;<br />
white-space: nowrap;<br />
visibility: hidden;<br />
display: inline-block;<br />
margin: 25px -9001px 0px -25px;<br />
opacity: 0;<br />
-moz-box-sizing: content-box;<br />
-webkit-transition: visibility 0s linear 0.1s,opacity 0.1s linear;<br />
-moz-transition: visibility 0s linear 0.1s,opacity 0.1s linear;<br />
-o-transition: visibility 0s linear 0.1s,opacity 0.1s linear;<br />
-ms-transition: visibility 0s linear 0.1s,opacity 0.1s linear;<br />
}<br />
<br />
div.author-desc {<br />
left: 0px;<br />
top: 0px;<br />
padding: 7px;<br />
background: #1A1A1A;<br />
border-radius: 5px;<br />
color: white;<br />
z-index: 0;<br />
height: 64px;<br />
}<br />
<br />
div.author-desc div {<br />
display: inline-block;<br />
}<br />
<br />
div.author-desc img {<br />
margin: 0px 7px 0px 0px;<br />
height: 50px;<br />
float: left;<br />
display:block;<br />
background: white;<br />
}<br />
<br />
div.author-hover:hover + div.author-hover-pic {<br />
visibility: visible; <br />
opacity: 1;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
-ms-transition: opacity 0.5s;<br />
}<br />
<br />
div.author-desc h5 {<br />
margin-top: 0px;<br />
text-align: left;<br />
color: white;<br />
}<br />
<br />
ul.side-nav li.nb-divider {<br />
margin-left: -30px;<br />
border-top: 1px solid #ddd;<br />
height: 0;<br />
padding: 0;<br />
}<br />
<br />
div.nb-week {<br />
display: none;<br />
border-bottom: none;<br />
}<br />
<br />
ul.side-nav li.nb-month {<br />
margin-left: -37px;<br />
border-top: 1px solid #ddd;<br />
height: 0;<br />
padding: 0;<br />
position: relative;<br />
}<br />
<br />
li.nb-month div {<br />
-webkit-transform: rotate(-90deg); <br />
-moz-transform: rotate(-90deg); <br />
-o-transform: rotate(-90deg); <br />
-ms-transform: rotate(-90deg);<br />
position: absolute;<br />
top: 47px;<br />
font-weight: bold;<br />
}<br />
<br />
div.nb-labels div {<br />
width: 16.66667%;<br />
float: left;<br />
text-align:center;<br />
font-size:14px;<br />
font-weight:bold;<br />
padding:0px;<br />
}<br />
<br />
div.nb-filter img {<br />
margin: 0px 0px;<br />
}<br />
<br />
div.filter-container {<br />
width: 100px;<br />
height: 100px;<br />
background-repeat: none;<br />
background-size: 100px 100px;<br />
cursor: pointer;<br />
-webkit-touch-callout: none;<br />
-webkit-user-select: none;<br />
-khtml-user-select: none;<br />
-moz-user-select: none;<br />
-ms-user-select: none;<br />
user-select: none;<br />
}<br />
<br />
div.filter-container img {<br />
height: 100px;<br />
}<br />
<br />
div.filter-container img:nth-child(2) {<br />
position: absolute;<br />
top: 0;<br />
}<br />
<br />
div#nb-alltech {<br />
float: right;<br />
}<br />
<br />
div.nb-onetech, div.nb-onetech-i {<br />
float: right;<br />
padding: 3px 10px 0px 10px;<br />
margin-top: -3px;<br />
cursor: pointer;<br />
}<br />
<br />
div.nb-onetech {<br />
color: #000000;<br />
background-color: #CACACA;<br />
border-top-left-radius: 10px;<br />
border-top-right-radius: 10px;<br />
}<br />
<br />
div.nb-onetech-disabled, div.nb-tech-disabled {<br />
display: none;<br />
}<br />
<br />
div.nb-onetech:after {<br />
content: ' \25BC';<br />
color: #000000;<br />
background-color: #CACACA;<br />
}<br />
<br />
div.nb-onetech-i {<br />
color: #8A8A8A;<br />
background-color: transparent;<br />
}<br />
<br />
div.nb-onetech-i:after {<br />
content: ' \25BA';<br />
color: #8A8A8A;<br />
background-color: transparent;<br />
}<br />
<br />
div.nb-tech {<br />
display: none;<br />
padding: 2px 5px;<br />
background-color: #CACACA;<br />
}<br />
<br />
div.nb-all, div.nb-all-i {<br />
font-family: 'Droid Sans', Helvetica, sans-serif;<br />
border-radius: 12px;<br />
text-align: center;<br />
font-weight: bold;<br />
padding: 3px 10px;<br />
display: inline;<br />
cursor: pointer;<br />
}<br />
<br />
div.nb-all {<br />
margin: 2px 25px;<br />
border: 0px solid;<br />
border-color: #F2F2F2;<br />
background-color: #505050;<br />
color: #F2F2F2;<br />
/* text-shadow: -1px -1px 0 #8A8A8A, 1px -1px 0 #8A8A8A, -1px 1px 0 #8A8A8A, 1px 1px 0 #8A8A8A; */<br />
}<br />
<br />
div.nb-all-i {<br />
margin: 2px 24px;<br />
border: 1px solid;<br />
border-color: #8A8A8A;<br />
background-color: #F2F2F2;<br />
color: #8A8A8A;<br />
/* text-shadow: -1px -1px 0 #8A8A8A, 1px -1px 0 #8A8A8A, -1px 1px 0 #8A8A8A, 1px 1px 0 #8A8A8A; */<br />
}<br />
<br />
div.nb-only {<br />
margin: 2px 25px;<br />
border: 1px solid;<br />
border-color: #F2F2F2;<br />
border-radius: 15px;<br />
text-align: center;<br />
font-weight: bold;<br />
color: #F2F2F2;<br />
cursor: pointer;<br />
/* text-shadow: -1px -1px 0 #8A8A8A, 1px -1px 0 #8A8A8A, -1px 1px 0 #8A8A8A, 1px 1px 0 #8A8A8A; */<br />
}<br />
<br />
div.nb-only-i {<br />
margin: 2px 25px;<br />
border: 1px solid;<br />
border-color: #8A8A8A;<br />
border-radius: 15px;<br />
text-align: center;<br />
font-weight: bold;<br />
color: #8A8A8A;<br />
background-color: #F2F2F2;<br />
cursor: pointer;<br />
/* text-shadow: -1px -1px 0 #8A8A8A, 1px -1px 0 #8A8A8A, -1px 1px 0 #8A8A8A, 1px 1px 0 #8A8A8A; */<br />
}<br />
<br />
div.nb-only, div.nb-only-i, div.nb-all, div.nb-all-i, div.nb-onetech, div.nb-onetech-i {<br />
-webkit-touch-callout: none;<br />
-webkit-user-select: none;<br />
-khtml-user-select: none;<br />
-moz-user-select: none;<br />
-ms-user-select: none;<br />
user-select: none;<br />
}<br />
<br />
div.entry {<br />
width: auto;<br />
max-width: none;<br />
min-width: 0;<br />
min-height: 50px;<br />
margin: 0 -15px;<br />
background-size: 50px 50px;<br />
background-repeat: no-repeat;<br />
margin-bottom: 10px;<br />
}<br />
<br />
div.entry:before, div.entry:after {<br />
content: "";<br />
display: table;<br />
}<br />
<br />
div.entry > div {<br />
padding-left: 65px;<br />
}<br />
<br />
div.entry p {<br />
margin: 0px;<br />
}<br />
<br />
div.entry div h6 {<br />
margin: 0px;<br />
font-weight: bold;<br />
}<br />
<br />
div.nb-wet { background-image:url('https://static.igem.org/mediawiki/2013/0/00/Cornell-nb-wet-tc.png'); }<br />
<br />
div.nb-dry { background-image:url('https://static.igem.org/mediawiki/2013/8/85/Cornell-nb-dry-tc.png'); }<br />
<br />
div.nb-model { background-image:url('https://static.igem.org/mediawiki/2013/5/50/Cornell-nb-model-tc.png'); }<br />
<br />
div.nb-hprac { background-image:url('https://static.igem.org/mediawiki/2013/c/c0/Cornell-nb-hprac-tc.png'); }<br />
<br />
div.nb-wiki { background-image:url('https://static.igem.org/mediawiki/2013/e/ed/Cornell-nb-wiki-tc.png'); }<br />
<br />
div.nb-anim { background-image:url('https://static.igem.org/mediawiki/2013/f/f6/Cornell-nb-pres-tc.png'); }<br />
<br />
div.nb-only#wet-only {<br />
background: #40B249;<br />
}<br />
<br />
div.nb-only#dry-only {<br />
background: #2D75B0;<br />
}<br />
<br />
div.nb-only#model-only {<br />
background: #AD2029;<br />
}<br />
<br />
div.nb-only#hprac-only {<br />
background: #E58A35;<br />
}<br />
<br />
div.nb-only#wiki-only {<br />
background: #5D3697;<br />
}<br />
<br />
div.nb-only#anim-only {<br />
background: #D5BA34;<br />
}<br />
<br />
ul.side-nav h6 {<br />
margin: 47px 0px 0px;<br />
}<br />
<br />
ul.side-nav.ntbk-nav h6 {<br />
margin: 87px 0px 0px;<br />
}<br />
<br />
table.check-off img {<br />
height: 20px;<br />
width: 20px;<br />
margin: auto;<br />
}<br />
/* -----------------------------------------<br />
Gallery Page<br />
----------------------------------------- */<br />
<br />
div.main-gallery ul {<br />
list-style: none;<br />
}<br />
<br />
div.main-gallery ul li {<br />
display: inline-block;<br />
margin: 0px 5px;<br />
}<br />
<br />
div.main-gallery ul li img {<br />
height: 80px;<br />
margin: 0px;<br />
}<br />
/* -----------------------------------------<br />
Functional Requirement Hover-overs<br />
----------------------------------------- */<br />
<br />
div.overflow {<br />
overflow: visible;<br />
}<br />
<br />
div.func-req-position {<br />
position: absolute;<br />
background: #e2e2e2;<br />
border-radius: 300px;<br />
width: 100px;<br />
height: 100px;<br />
background: #f2f2f2;<br />
color: white;<br />
opacity: 1;<br />
}<br />
<br />
div.func-req-hover {<br />
width: 100px;<br />
border-radius: 300px;<br />
}<br />
<br />
div.func-req-hover img {<br />
height: 100px;<br />
margin-top: -4px;<br />
opacity: 0.5;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.func-req-hover:hover img {<br />
opacity: 1;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.func-req-desc {<br />
position: absolute;<br />
left: -75px;<br />
top: 100px;<br />
width: 250px;<br />
padding: 7px;<br />
background: #1A1A1A;<br />
border-radius: 5px;<br />
color: white;<br />
opacity: 0;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.func-req-hover:hover + div.func-req-desc {<br />
opacity: 1;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.func-req-desc h5 {<br />
text-align: center;<br />
color: white;<br />
}<br />
<br />
/* -----------------------------------------<br />
Device Hover-overs<br />
----------------------------------------- */<br />
<br />
div.device-position {<br />
position: absolute;<br />
border-radius: 300px;<br />
width: 55px;<br />
height: 55px;<br />
opacity: 1;<br />
}<br />
<br />
div.device-hover {<br />
position: absolute;<br />
width: 55px;<br />
height: 55px;<br />
z-index: 5;<br />
border-radius: 300px;<br />
}<br />
<br />
div.device-hover-pic {<br />
width: 783px;<br />
height: 489px;<br />
z-index: 3;<br />
opacity: 0;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.device-hover-pic img {<br />
width: 783px;<br />
height: 489px;<br />
margin: 0px;<br />
opacity: 1;<br />
}<br />
<br />
div.device-desc {<br />
position: absolute;<br />
left: 10px;<br />
top: 350px;<br />
width: 170px;<br />
padding: 7px;<br />
background: #1A1A1A;<br />
border-radius: 5px;<br />
color: white;<br />
z-index: 4;<br />
}<br />
<br />
div.device-hover:hover + div.device-hover-pic {<br />
opacity: 0.9;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.device-hover.total:hover + div.device-hover-pic {<br />
opacity: 1;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.device-desc h5 {<br />
margin-top: 0px;<br />
text-align: center;<br />
color: white;<br />
}<br />
<br />
/* -----------------------------------------<br />
Circle Hover-overs<br />
----------------------------------------- */<br />
<br />
div.circle-position {<br />
position: absolute;<br />
border-radius: 300px;<br />
width: 55px;<br />
height: 55px;<br />
opacity: 1;<br />
}<br />
<br />
div.circle-hover {<br />
position: absolute;<br />
width: 250px;<br />
height: 250px;<br />
z-index: 5;<br />
border-radius: 300px;<br />
}<br />
<br />
div.circle-hover-pic {<br />
width: 783px;<br />
height: 760px;<br />
z-index: 3;<br />
opacity: 0;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.circle-hover-pic img {<br />
width: 783px;<br />
margin: 0px;<br />
opacity: 1;<br />
}<br />
<br />
div.circle-desc {<br />
position: absolute;<br />
left: 307px;<br />
top: 343px;<br />
width: 170px;<br />
padding: 7px;<br />
background: #1A1A1A;<br />
border-radius: 5px;<br />
color: white;<br />
z-index: 4;<br />
}<br />
<br />
div.circle-hover:hover + div.circle-hover-pic {<br />
opacity: 1;<br />
-webkit-transition: opacity 0.5s;<br />
-moz-transition: opacity 0.5s;<br />
-o-transition: opacity 0.5s;<br />
}<br />
<br />
div.circle-desc h5 {<br />
margin-top: 0px;<br />
text-align: center;<br />
color: white;<br />
}<br />
<br />
/* -----------------------------------------<br />
Override styling for badge drop-downs<br />
----------------------------------------- */<br />
div.badge-wrapper-main, div.badge-wrapper {<br />
position: absolute;<br />
background: #f2f2f2;<br />
width: 80px;<br />
height: 80px;<br />
border-radius: 80px;<br />
border: 10px solid #b31b1b;<br />
}<br />
<br />
div.badge-wrapper-main {<br />
top: -20px;<br />
left: 0px;<br />
float: right;<br />
}<br />
<br />
div.badge-wrapper {<br />
left: auto;<br />
right: -32px;<br />
margin: 10px;<br />
margin-top: -9px !important;<br />
}<br />
<br />
div.badge-wrapper a {<br />
background: none !important;<br />
border: none !important;<br />
padding: 0px !important;<br />
-webkit-box-shadow: none !important;<br />
-moz-box-shadow: none !important;<br />
box-shadow: none !important;<br />
}<br />
<br />
div.badge-wrapper+a {<br />
margin-top: 19px;<br />
padding-left: 15px !important;<br />
font-size: 24px !important;<br />
background: #f2f2f2 !important;<br />
width: 290px;<br />
margin-left: -40px;<br />
border: 5px solid #b31b1b !important;<br />
}<br />
div.badge-wrapper:hover+a, div.badge-wrapper+a:hover {<br />
background: #d2d2d2 !important;<br />
}</div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/notebookTeam:Cornell/notebook2013-09-28T01:51:12Z<p>R.Lizarralde: </p>
<hr />
<div><!-- paulirish.com/2008/conditional-stylesheets-vs-css-hacks-answer-neither/ -->{{:Team:Cornell/templates/header}}{{:Team:Cornell/templates/notebook}}<br />
<html class="no-js" lang="en"><br />
<!-- -------------------Notebook entries------------------- --><br />
<div id="weekAentry" class="nb-week" style="display: block"><br />
<div class="twelve columns"><br />
<h3 class="centered">January</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 5th<br />
|text=Applications for new Cornell iGEM members were due today-- we've got 57!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 16th<br />
|text=After an eternity of reading and a gigantic spreadsheet of evaluations, we're finally ready to start interviewing applicants-- 36 of them!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 25th<br />
|text=I'm really excited about having these new members on the team. There were many awesome interviews, but now we have to sit down and make hard decisions on who to take, as most of last year's members are graduating, so there's a limit to how many new members we can train. This is going to be a tough weekend.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 27th<br />
|text=After several hours of discussion, we decided on our 21 new members! Congratulations, Tim Abbott, Hannah Ajmani, Eric Appel, Ryan Ashley, Nupur Bhatt, Arun Chakravorty, Rebecca Chew, Sharlene Dong, Sara Gregg, Alex Han, Eric Holmes, Daniel Leach, Oat Luengvarinkul, Jeffrey Ly, Ritvik Sarkar, Mac Sennett, Prashant Sharma, Olya Spassibojko, Yoshiko Toyoda, and Kyle Wheeler!<br />
|author=Rafael<br />
}}<br />
<html> <br />
</div><br />
</div><br />
<div id="weekBentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">February</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 1st<br />
|text=We had our first full team meeting today with all the new members, introducing them to the iGEM competition, and did an icebreaker we call speed-taboodunit! For the next week, they have the assignment of analyzing a previous iGEM project (in groups), and explaining what the project was trying to accomplish, whether synthetic biology was a good approach for this, and reasons for their success or lack thereof.<br />
|tech=Speed-taboodunit is a mashup of speed-dating, taboo, and whodunit; the idea is that each person writes on a piece of paper something unusual about themselves, underlining the key words, then these pieces of paper are randomly redistributed. Everyone organizes in a speed-dating format, with two lines of people, and you have a minute to talk to them and try to find out if they're the person described on their piece of paper-- with the catch that they can't use any of the underlined words. People who successfully find their person leave the group, but otherwise after the minute everyone moves down the line. As for why we came up with this specific icebreaker, we wanted to hit two key requirements of icebreakers: they have to be fun, and they have to make specific accommodations for being shy or quiet. Because each person has the specific objective of finding their person, this helps them overcome the shyness, and as for the fun, everyone loves taboo!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 7th<br />
|text=Our first social event tonight went well! Hopefully with a few more of these we'll have some great team comradery and feel more comfortable around each other. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 9th<br />
|text=At our second team meeting we went over the past project analyses, as well as some biology review-- mostly molecular cloning. For the next week, we've assigned more past projects for them to outline in more detail.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 16th<br />
|text=Today we went over literature review, went over the past project outlines, and gave a two-week problem-oriented brainstorming and project outline assignment. Now that each member has looked at several past projects and analyzed them, we're moving into brainstorming for this year's project and practicing the development of project pitches.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=February 21th<br />
|text=We just started working on the wiki! Our first assignment: figuring out how wiki templates work. With templates the website would become much easier to edit, but for some reason HTML and wiki text don't seem to work very well together...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 23th<br />
|text=For the second week of this assignment, we compiled the brainstormed ideas and had members pick which ones they wanted to explore further. We also had a movie night planned, but it fell through :(<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=February 28th<br />
|text=Finally got templates to work! The trick was ending HTML right before wiki text, and starting HTML again right afterwards. Not very elegant, but it works.<br />
|author=Nupur<br />
}}<br />
<html> <br />
</div> <br />
</div><br />
<div id="weekCentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">March</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 1st<br />
|text=We want to revamp the notebook page with a filter system that lets you sort by subteam. We set up the basic layout of the page, but writing the javascript is going to be another story.<br />
|tech=Check out our <html><a href=https://2012.igem.org/wiki/index.php?title=Team:Cornell/Sandbox/notebook&oldid=299789 target="_blank"> notebook</a></html> before anything worked!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 2nd<br />
|text=Groups over the previous week investigated phage therapy, piezoelectric bacteria, "sticky paper", logic gates / bistable switches, phytases, mycelia, and pressure-adaptive materials. We're keeping the results from those, but doing a second round of brainstorming over the next few weeks-- the idea is to allow the exploration of any ideas that arose while researching the previous round of ideas.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 5th<br />
|text=We just finished the javascript for filtering. Rafael also made cool subteam icons for the filter buttons.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 6th<br />
|text=I made a template for notebook entries and added styling to each entry.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 9th<br />
|text=We compiled our second round of ideas, including topics like methanotrophs, a CRISPR knockout system, and suppressor tRNA inducible expression systems. We also took our first team picture, and probably the only one that will have all of last year's and this year's members together!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 29rd<br />
|text=Spring break knocked out a couple of meetings, but it looks like some research was accomplished nonetheless! The phytase project idea is looking pretty promising-- it incorporates a lot of different disciplines, with stuff for our mechanical and electrical engineers to do, as well as modeling.<br />
|tech=A lot of iGEM teams we've looked at don't have much beyond the biology, so we really try hard to do interdisciplinary projects, as they provide a great opportunity for learning to work with different kinds of people.<br />
|author=Rafael<br />
}}<br />
<html> <br />
</div> <br />
</div><br />
<div id="weekDentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">April</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 6th<br />
|text=We've narrowed it down to four project ideas: smokestack reactors (capturing pollutants with bioreactors), incorporation of phytases into anaerobic digesters, mycelial materials, and using bacteria to address issues with artificial implantable kidneys.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 10th<br />
|text=We had a call today with Ecovative Design, a company that uses mushrooms to make a biodegradable styrofoam analogue-- our mycelial materials project idea would involve working with them to improve their product. It seems like our interests are aligned and that we should be able to work with them. Exciting! <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 13th<br />
|text=This meeting was dedicated to project pitches from each of the four ideas, followed by voting on which project we should pursue. Some of our graduate advisors also attended to provide a more seasoned perspective on the viability of each project. After some deliberation and spreadsheet magic, we decided on mycelial materials!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 20th<br />
|text=Today we worked on team structure for the summer; Swati will be the overall team leader, with me and Mark coordinating wetlab, Olya taking meeting minutes, Mac running drylab, and Hannah will be managing human practices. Later on in the summer, we will also develop task groups. For the next week, we've also split into groups to research various aspects of the mycelial materials project.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 26th<br />
|text=We've compiled initial research on carotenoid pigments, transformation protocols, DNA that may be useful for a basidiomycete toolkit, and a few other topics. Over the next week we'll meet with several professors on campus who work with fungi to see what resources and advice they can provide.<br />
|author=Rafael<br />
}}<br />
<html> <br />
</div> <br />
</div><br />
<div id="weekEentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">May</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=May 4th<br />
|text=We met with Professor Gillian Turgeon, who does fungal transformation with Cochliobolus heterostrophus, an ascomycotic corn pathogen. We can get a few of the genes we've been looking for from her, yay! We also met with Professor Teresa Pawlowska, who directed us towards some other fungi and resources for fungi, as well as provided some background on fungal biology. As for the rest of the semester, we're letting everyone off the hook-- we don't want anyone to fail their exams because of iGEM!<br />
|author=Rafael<br />
}}<br />
<html> <br />
</div><br />
</div><br />
<div id="week1entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered" >Week 1</h3><br />
<h5 class="centered">(06/17 - 06/23)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 17th<br />
|text=The drylab team met briefly on June 17 to begin discussing some ideas. We went over some of the growing methods for Ganoderma lucidum, brainstormed some non-packaging related ideas for a fungal biomaterial, and discussed how we could test some of the physical characteristics of the final product. We thought it might be a good idea to test the thermal conductivity if it were to be used as a drinking cup or something. We also determined that people probably do not want to drink out of a fungus cup. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 18th<br />
|text=We started bootcamp today, doing our first transformations with kit plate biobricks.<br />
|tech=We transformed parts containing carotenoid pathway genes ''crtE'', ''crtI'', ''crtB'', and ''crtY'' (BBa_K523022, BBa_K539119, BBa_K145001), as well as the T7 promoter (both in pIVEX2.3d as well as BBa_I712074), the T7 polymerase gene (BBa_K145001), and the transformation efficiency kit part (BBa_J04450).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 19th<br />
|text=The second day of bootcamp had more activity-- we PCR-amplified a variety of parts to biobrick them and get ready for making composite parts later.<br />
|tech=From the plasmid pUCATPH (pAh13G), we amplified the ''trpC'' promoter (P''trpC'', a constitutive fungal promoter) and the ''trpC'' terminator (T''trpC''). From pNG (pAg13H), we amplified ''nptII'', the geneticin (a plant/fungal antibiotic) resistance gene.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 20th<br />
|text=I finally started moving pages to the 2013 wiki! It's looking very empty right now, but Rafael made a gif of our logo with his mycodraw program to use as a homepage placeholder. We also decided to add a technical details section to each of our notebook entries so that we can separate the jargon from daily summaries.<br />
|tech=Check out the <html><a href=https://static.igem.org/mediawiki/2013/2/21/CornellStillGrowing.gif target="_blank">gif</a></html> if it's not on our homepage anymore. Originally Rafael converted his mycodraw program from java to javascript (which took a VERY long time), but the script only worked on some browsers :( We had to settle for the gif instead.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 20th<br />
|text=On our third day of bootcamp, we taught new members how to run gels, do minipreps, and run digests, continuing our preparation of biobrick parts from the previous two days.<br />
|tech=We miniprepped the ''crtEIB'' (BBa_K523022), ''crtY'' (BBa_K539119), and T7 (BBa_I712074) biobricks. The gels confirmed our PCRs for P''trpC'', T''trpC'', and ''nptII'', which we then digested to insert into pSB1C3.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 21st<br />
|text=The javascript for toggling technical details took a bit longer than expected, but we finished it up today.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 21st<br />
|text=Continuing with bootcamp, we column-purified the digests of PCR products and gel-purified the digested pSB1C3 backbone, dephosphorylated, and ligated, resulting in our first batch of biobricks or pre-biobrick parts.<br />
|tech=P''trpC'', T''trpC'', and ''nptII'' were column-purified, digested pSB1C3 backbone was gel-purified and dephosphorylated, and each was ligated into the pSB1C3 backbone.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 22nd<br />
|text=I added a flyout to notebook entries authors with a picture, short description, and link to their bio on the team page.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 22nd<br />
|text=Our penultimate day of bootcamp was a light one-- we only transformed cells with the constructs we'd made over the past four days. <br />
|tech=We transformed T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) constructs into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 23rd<br />
|text=We concluded bootcamp with colony PCRs to test whether each of the constructs was present. Unfortunately, the gels had no bands, so we grew overnight cultures and will screen the minipreps instead.<br />
|tech=We used Q5 PCRs with the standard biobrick primers VF2 and VR to attempt to confirm the presence of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R).<br />
|author=Rafael<br />
}}<br />
<html> <br />
</div><br />
</div><br />
<div id="week2entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 2</h3><br />
<h5 class="centered">(06/24 - 06/30)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 24th<br />
|text=The drylab team met to update each other on some of the preliminary research. The main focus of this meeting was the development of a growing protocol. Sara found a professor that we could contact in the material science department for help regarding the testing of material properties. Rebecca and Manny looked into what different aspects of the fungi we could model, such as growth, decay, etc. Ritvik brainstormed some more ideas regarding the potential uses of this material, and came up with the idea of using it as insulation. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 24th<br />
|text=I started working on a hypertexting script that will automatically recognize words on a page and add a flyout with a description. We can use this to recognize names for the author flyouts I implemented yesterday, as well as other keywords such as plasmid names and protocols.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 24th<br />
|text=We miniprepped our three constructs. Then we went back to sleep.<br />
|tech=Minipreps of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 25th<br />
|text=I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 25th<br />
|text=We did another PCR from the miniprepped plasmids to confirm the right sizes of inserts, then sent them in for sequencing.<br />
|tech=The Q5 PCRs of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) showed bands of the correct size, so sequencing with VF2 and VR was submitted to make sure nothing strange had happened.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 26th<br />
|text=The script is now working on all browsers except for Internet Explorer.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 27th<br />
|text=We started cloning crt genes downsteam of the T7 promoter. Because the T7 promoter is too small to extract as a fragment, we're placing crt genes downstream, then transferring the promoter+gene fragments back to pSB1C3. Today we digested ''crtY'' and the T7 promoter plasmid, but the digestion of ''crtY'' failed. <br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). However, the gel showed only one band for each (what one would expect for pAK13D, but pC13B should have had two bands).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 28th<br />
|text=We redid the digestion of ''crtY'' and the T7 promoter plasmid from yesterday, apparently successfully this time. We also started liquid cultures of pBARGPE1, a plasmid we got from the Fungal Genetics Stock Center.<br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). The gel this time had two bands at the correct locations for pC13B, so we're hoping it's a success this time. We also started overnight LB cultures of pAb13T (pBARGPE1).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 29th<br />
|text=Crossing our fingers, we gel extracted and ligated the ostensibly successful digests of ''crtY'' and the T7 promoter plasmid, and then transformed. We also digested ''crtI'' and ''crtB'', other carotenoid genes that we will also be putting under the T7 promoter, and miniprepped the pBARGPE1 that had been grown up last night.<br />
|tech=If successful, the ligation will form pAK13AD. pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI for insertion into pAK13D, and separated on a gel. pAb13T (pBARGPE1) was miniprepped and quantified by nanodrop. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 30th<br />
|text=Unfortunately, there were no colonies on the transformation plate for T7+''crtY'', so we redid the digestions for those. We also digested T''trpC'' and ''nptII'' for the eventual cloning of a P''trpC''+''nptII''+T''trpC'' construct (we have to put the parts together in reverse because ''nptII'' contains a PstI site, which we will be trying to remove as well). Gel extractions of digested ''crtI'' and ''crtB'' were also empty (insignificant concentrations), so these digestions were also redone, and all were run on a gel, which looked mostly okay.<br />
|tech=pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (T''trpC'') with EcoRI and XbaI, and pCg13R (''nptII'') with EcoRI and SpeI, and all were separated on a gel; ''crtI'' looked faint, and pAK13D was smeared, but all fragments were cut out for purification tomorrow.<br />
|author=Rafael<br />
}}<br />
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<div id="week3entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 3</h3><br />
<h5 class="centered">(07/01 - 07/07)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 1st<br />
|text=We finished the hypertexting script and fixed some miscellaneous problems. I think we can call this a wrap. The notebook page is officially done!<br />
|tech=Now we just have to start filling out entries...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 1st<br />
|text= Two of our team members participated in SILS Skills Night on June 20th.<br />
|tech= Price of one 12 oz Styrofoam cup: 4.09 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 1st<br />
|text=Yesterday's digestions were gel extracted, ligated, and transformed. Hopefully it worked this time!<br />
|tech=Digested pAK13D was dephosphorylated, then ''crtI'' , ''crtB'', and ''crtY'' were ligated with pAK13D to put them under the T7 promoter, hopefully producing pAK13AB, pAK13AC, and pAK13AD, respectively. pCg13R was also ligated with pC13Q, hopefully producing pCg13U. All four constructs were transformed into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 2nd<br />
|text=There were no transformants, again :(. On the other hand, we received our tube of ''Ganoderma lucidum'' from Dr. David Hibbett today! Once more with feeling, we redid the four digestion-ligations for ''crtI'', ''crtB'', ''crtY'', and ''nptII''. We also amplified ''hph'', the hygromycin resistance gene, from one of the plasmids we got from Dr. Turgeon's lab, as well as pSB1C3 backbone, and we will use Gibson assembly to put them together (as ''hph'' has both EcoRI and PstI sites)-- the gels looked good for both of these. Finally, we submitted pAb13T (pBARGPE1) for sequencing to determine the sequences of the ''Aspergillus nidulans'' ''gpdA'' promoter and the ''bar'' gene, which confers phosphonothricin resistance.<br />
|tech=''hph'' was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 3rd<br />
|text=All transformations failed yet again, and were repeated once more. Four tears shed for the lost. ''hph'' was Gibson-assembled, and we ran a Q5 PCR of the ''bar'' gene, under the assumption it matches other published ''bar'' sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on ''nptII'' (pCg13R) to remove its internal PstI site.<br />
|tech=:( ;_; T_T D: (tears for the transformations lost). The ''bar'' PCR and pC13F were digested with EcoRI and PstI, and ligated to hopefully form pCp13X. Site-directed mutagenesis was performed both with and without DpnI to digest template DNA. <add something about site-directed mutagenesis design; refer to primers? probably requires Swatinput><br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 5th<br />
|text=Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.<br />
|author=Nupur<br />
}}<br />
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<div id="week4entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 4</h3><br />
<h5 class="centered">(07/08 - 07/14)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 10th<br />
|text= Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 11th<br />
|text=After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop. <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 11th<br />
|text=Digest screens were run to see if cloning had been successful,, and Gibson cloning was continued. <br />
|tech=Minipreps were made from the ''crtY'' plates, from which a digest screen was prepared using ''Not-I'' to see if the ligation worked. The digestions were run on a gel and some appear to have worked (X/P/S, X/P/A #2/3, and X/P/S/A #⅔ worked). Also, a glycerol stock of ''nptII BB'' was made and miniprepped, but the concentration was extremely low, so an overnight culture was made from the glycerol stock to be miniprepped tomorrow. Another transformation was done using the remaining miniprep of ''nptII BB'' from earlier. Cultures were made for ''Gibson #3'', and a colony PCR perfomed, then run on a gel. Non-GC fragments from Gibson #3 were too small, but GC #⅖ were good (1kb fragment), and #4 had a band at 1kb with another smaller fragment. Finally, ''nptII BB'' was digested with X/P then column purified, and ''PtrpC'' was digested with S/P then gel purified.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 12th<br />
|text=Cloning with ''nptII BB'' and ''PtrpC'' was continued, as well as cloning with the Gibson Assembly method.<br />
|tech=Today ''nptII BB'' and ''PtrpC'' were quantified. Also, ''nptII BB'' transformants were miniprepped, and another glycerol stock of ''nptII BB'' was made. The DNA for Gibson Assembly was miniprepped, and GC colonies 2,4, and 5 from the Gibson #3 gel yesterday were prepared for sequencing. ''NptII BB'' was digested with the ''PtrpC'' digest from yesterday. More cultures of X/P/S #1/2/3, X/P/A #⅔, and X/P/S/A #⅔ were made. CYM medium was created, and ''PC13Z'' was transformed from a kit plate.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 13th<br />
|text=Today we worked with fungi! We applied different amounts of antibiotic to G. lucidum, to test the sensitivity of our strain of fungi. In addition, cloning of ''pCg13Y'' was nearly completed. <br />
|tech=''pCg13Y'' was desalted, transformed, then plated into E. coli. Antibiotic stocks ''G418'' and ''PPT'' were made (100x). G. lucidum cultures were inoculated with varying levels of antibiotic to test the strain's sensitivity for future selection purposes. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 14th<br />
|text=Our cloning of ''pCg13Y'' was found to be unsuccessful. ''pC13z'' and ''pAK13AD'' were prepped for cloning. <br />
|tech= Colony PCRs and cultures of ''pCg13Y'' were completed to determine the success of cloning ''nptII BB'' downstream of ''PtrpC''. Unfortunately, a gel revealed that the dna fragments appeared at 0.6kb instead of 1.4kb. ''pC13Z'' and ''pAK13AD'' were miniprepped, but had to be thrown out due to contamination of our EB. New cultures were made to be miniprepped again. A glycerol stock of ''PC13Z'' was made, too. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 14th<br />
|text=This week, drylab split up so members could focus on their strengths. Rebecca and Sara began designing our feedback controller, with some much appreciated advice from the previous drylab team leader, Dan. Ritvik, Manny, and Mac began coming up with design considerations for the incubation chamber. We were most concerned with size; too small and it wouldn't be useful, too big and it would be difficult to control any variables. We eventually decided that a 1x1x2' chamber would probably be a good compromise. Rebecca, Sara, Ritvik, and Mac also attended machine shop training this week. In order to complete our training, we each had to construct a small aluminum lamp using a milling machine and a lathe. Fortunately, everyone emerged from the training session one lamp richer, and with a full set of fingers on each hand.<br />
|author=Mac <br />
}}<br />
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<div id="week5entry" class="nb-week"><br />
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<h3 class="centered">Week 5</h3><br />
<h5 class="centered">(07/15 - 07/21)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 15th<br />
|text=Rebecca looked into the components of the temperature feedback control; namely, the temperature sensor, the microcontroller and the heating element. Put in an order for the temperature sensor, microcontroller (an Arduino Fio) and Xbee ports for wireless communications.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 15th<br />
|text=Gibson ''hph'' was submitted for sequencing, and cloning of ''crtY'' and ''crtB'' was continued. <br />
|tech=Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. Gibson ''hph'' was submitted for sequencing, and ''pAK13AD''s and ''pC13Z''(GFP) were miniprepped. ''PC13Z'', ''PC13F'', ''PCP13X'', ''PC13M'', and ''PC13E'' were PCRed with primers 31 and 32, the standard ''pSB1C3'' primers. A gel was run to check the PCRs-all looked successful except ''pC13M'' and ''pC13E'' which had nonspecific bands around 1kb. A new technique was used in the following digests, which used a ''restriction cocktail'' of enzymes to cut the dna. ''PAK13AD'' was digested with E/S/SacI/ClaI so that it could be placed in ''PSB1C3'' using ''SpeI'' instead of ''PstI'' due to the small distance between the ''PstI'' and the ''SacI'' cut sites. ''PC13F'' was digested with E/S/KpnI so ''PAK13AD'' can be placed in it. ''PAK13D'' was digested with S/P so ''crtI''/''crtB'' can be placed in it. All the digests were then column purified rather than gel purified--a move which has improved our ligation efficiencies. Then the purifications were quantified. Cultures were made for ''PC13P'' and ''PC13K''. Finally, ''PAK13AD'' was dephosphorylated then ligated into ''PC13F'' (''PC13AH''). <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 16th<br />
|text=One iGEM member spoke at a panel for pre-freshmen biology students interested in pursuing research positions in college (PSP).<br />
|tech= Price of one 12 oz Styrofoam cup: 4.95 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 16th<br />
|text=<br />
|tech=Sequencing results came back positive for the Gibson cloning of hph, so a culture of gibson colony 5 was made. ''PC13AH'' was transformed and plated, and ''pC13K'' and ''pC13K'' were miniprepped. ''pCp13X'' (S/P), ''PC13F'' (S/P), ''PC13Z'' (S/P) were purified and digested along with ''PC13P'' (X/P) [also dephosphorylated], then all were column purified. Reran a PCR of ''PC13M'' and ''PC13E'', and one of ''PC13K'' with primers 31/32. All PCRs were cleaned, then run on a gel along with ''PBARGPE1'' and ''PBARGPE1'' which had been digested with NotI today. Results: ''PC13E'' had a 3kb fragment (good), ''PC13K'' had a .9kb fragment (should be 1.7-1.8kb), ''PC13M'' had a 1.2-1.5kb fragment (good), ''pBARGPE1'' had a 4kb fragment (super-coiled) and ''pBARGPE1'' with NotI had a 5-6kb fragment (has a NotI site-good). From these results it can be concluded that everything worked except ''PC13K''. Ligations of ''PAK13D'' to inserts ''PC13Z'', ''pCp13X'', and ''PC13F'' along with vector ''PC13P'' to inserts ''PC13Z'', ''pCp13X'', and ''PC13F'' were performed. Also, overnight digestions of ''PC13M'', ''PC13E'', and ''PC13K'' with X/P were set up. Transformation was attempted for ''PC13AI'' and ''PA13AJ'', but the cuvettes weren't cleaned properly so it failed. Finally, our dwindling supplies prompted the ordering of more Spe1 and membrane filters. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 18th<br />
|text=The restriction cocktail method of cloning worked! Cloning of pC13AH (''T7'' + ''crtY'') was shown to work using this method. We are also continuing our cloning of the ''crtI'' and ''crtB'' pathways. <br />
|tech=All 18 (yes, 18!) of the ''pC13AH'' colonies that had been screened by colony PCR yesterday were miniprepped. We digested the miniprepped colonies of pC13AH clones (T7 promoter with crtY in pSB1C3) with ''NotI'' to see if the restriction cocktail method of cloning worked. On the gel that was subsequently run, bands were expected at ~2.1 for pSB1C3 and ~1.2 for ''crtY''+''pT7''. The restriction cocktail method worked! The bands appeared at the correct lengths on the gel for ''pC13AH''. There was not enough miniprepped DNA for pC13AH to send for sequencing, so the colonies were cultured again for another miniprep. None of the ligations into pAK13D (pSB1AK8 + PT7) worked -- pC13Z (''GFP''), pC13M (''crtB''), and pCp13X (''bar'') all failed => try cloning only using restriction cocktail method, instead of attempting PCR with VF2 & VR. Digested pC13Z, pC13M, pCp13X, pC13K (''crtI'') with XbaI and PstI in addition to ApaLI and SacI for later ligation with pAK13. pAK13D had been previously digested with SpeI and PstI, column purified, and dephosphorylated for insert ligation. More pAK13D was grown to acquire more DNA to be miniprepped tomorrow. Nupur performed colony PCR and made cultures of transformants from yesterday. All transformations appeared to have very low efficiency--perhaps an issue with heat shock stocks? Transformations of ''AI-AL'' (mRFP cds and lox site parts) all yielded colonies, though at really low efficiencies compared to most transformations off of kit plates. These were screened by colony PCR to verify. Ligations into pC13P all yielded colonies, but at very low efficiencies. These were pC13E (T7 pol), pCp13X (bar), and pC13Z. All were screened by colony PCR as well. Taq was used to perform 5 colony PCRs, then all colony PCRs were run on a gel. Also, pCg13s (nptIIBB)was digested with XbaI, PstI, ApaLI, and SacI for later ligation to pC13P, which had been previously digested with SpeI and PstI, column purified, and dephosphorylated. Overnight ligations of pC13Z, pC13M, pCp13X, pC13K, pCg13S with vector pAK13D were set up, along with overnight ligations of pC13K and pCg13S in vector pC13P. <br />
|author=Danielle<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 21st<br />
|text=This week, the fabrication group focused on what materials to construct our chamber from. At first we considered a single wall design, but this was eventually scrapped, as we were unable to find any material that would provide sufficient insulation at a reasonable thickness (and cost). After putting our heads together, we decided on a double wall with an insulating layer in between them. Mac began drawing up the chamber in Solidworks. <br />
|author=Mac <br />
}}<br />
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<div id="week6entry" class="nb-week""><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 6</h3><br />
<h5 class="centered">(07/22 - 07/28)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 23rd<br />
|text=The parts have arrived and Rebecca configured them. Met up with Paras who was on last year's team to get a detailed explanation of how their project had worked as well.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 23rd<br />
|text=Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 28th<br />
|text=This week, the fabrication group finalized the incubator design and ordered the construction materials! We decided to construct the outer and inner layers out of HDPE plastic in the rather lab appropriate color of white. For our insulation, we decided to use high density polystyrene. After comparing the K-factors for various types of insulation, we determined that this material gave us the most bang for our buck, so to speak. <br />
|author=Mac <br />
}}<br />
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<div id="week7entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 7</h3><br />
<h5 class="centered">(07/29 - 08/04)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 31st<br />
|text=Sara and Rebecca met up with our advisor Dr Bruce Land and he provided lots of helpful advice with regard to the design considerations (where the heater should be in our incubation chamber, convection currents, type of heating plate to use). He also suggested that we use a proportional-integral-derivative controller (PID controller) to regulate the heating.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 1st<br />
|text= Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 2nd<br />
|text= Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 4th<br />
|text=Mac went to visit Ecovative Design with Swati, Rafael, Alex, and Hannah. We were able to meet with Gavin McIntyre, one of the founders of the company, who was kind enough to give us a tour of their facilities. He also gave our wetlab members some pointers for our own project. In other news, we are still waiting for our materials to arrive. <br />
|author=Mac <br />
}} <br />
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<div id="week8entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 8</h3><br />
<h5 class="centered">(08/05 - 08/11)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 5th<br />
|text= Eric Holmes and Arun Chakravorty presented for a group of scientific researchers (SILS)<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=August 7th<br />
|text=We helped [[Team:Ciencias-UNAM|Ciencias-UNAM]] get set to transfer their HTML website to the wiki. Hopefully we'll get up a tutorial for what to look out for on here soon!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 7th<br />
|text=The sensor has been connected! <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 10th<br />
|text= Five team members presented at the Ithaca Sustainability Center for citizens of Tompkins County.<br />
|author=Hannah<br />
}}<br />
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<div id="week9entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 9</h3><br />
<h5 class="centered">(08/12 - 08/18)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 15th<br />
|text=Rebecca looked more into the details of the pulse-width modulation and PID controller so that the heater can be turned on for certain periods of time for more even heating. <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=August 18<br />
|text=Today, the dastardly trio of Arun, Hannah, and Eric were chosen to present at the regional iGEM competition!<br />
|tech=<br />
|author=Swati<br />
}}<br />
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<div id="week10entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 10</h3><br />
<h5 class="centered">(08/19 - 08/25)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 25th<br />
|text=Christine's back! She started looking at the components for a humidity sensor and placed an order for a mistmaker which would be able to control humidity levels. In the meantime, Sara studied the details of the heating circuit which utilizes a transistor to amplify signals and switches it such that the resistor could be used as the heating element. <br />
|author=Rebecca<br />
}}<br />
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<div id="week11entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 11</h3><br />
<h5 class="centered">(08/26 - 09/01)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 26th<br />
|text=Our materials are here and everyone in the fabrication group has returned to Ithaca! This week, Manny and Mac focused their efforts on getting the plastic cut to the proper dimensions. Unfortunately, this proved more to be more difficult than anyone could have imagined. After transporting a rather hefty 48"x48" piece of plastic across campus from Weill Hall to the Emerson Lab machine shop, we were told that we would have to take it elsewhere to have it cut (plastic is bad for blades). Disappointed, but never defeated, we decided to try our luck with another machine shop. Also, carrying plastic around campus isn't much fun, so we held it on the roof of a car instead (not recommended). Fortunately, no traffic violations were received, and the Clark hall machine shop was more than happy to cut up our plastic for us. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 1st<br />
|text=Lydia's back too! Discussed what we have been doing for the past few weeks and immediate goals. <br />
|author=Rebecca<br />
}}<br />
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<div id="week12entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 12</h3><br />
<h5 class="centered">(09/02 - 09/08)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 3rd<br />
|text=Christine, Rebecca and Sara met up with Dr. Land again and we learnt more about setting up PID controls. Went to the lab next where Christine and Sara set up the heating element circuit, Rebecca soldered pins onto the arduino and Lydia drew up the circuit design.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type= anim<br />
|head=September 6th <br />
|text= We met at Flora Rose house to decide on an outline of the presentation and also decide a timeline.We met for the first time to discuss a preliminary outline for the presentation. Hannah will be handling all the human practices elements, Eric will be explaining relevant biological background and strategies, and Arun will be presenting all of our characterization data (hopefully!). We made a detailed structure which will include an introduction about styrofoam pollution and ecovative. Then we will move on to a brief overview of our wetlab work. It was clear that the wetlab portion should be divided into 3 main components. The components would be fungal toolkit, carotenoid pathway, and antifungals. Finally, we will end with results, human practices, and acknowledgments to all our beautiful advisors and sponsors.<br />
|tech= <br />
|author=Arun<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=September 7th<br />
|text= Five team members went to the Ithaca Sciencenter and taught a group of 18 children and 17 parents about the power of DNA.<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type= anim<br />
|head=September 7th <br />
|text= After researching we found out that the powerpoint presentation should be centered around design and images rather than content. We decided to meet tomorrow to discuss possible design layouts and schemes. We looked at many examples of presentations and considering our project, we think we should have a very modern and green theme. Arun will explore with this later on.<br />
|tech= <br />
|author=Arun<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 8th<br />
|text=We met super early in the morning (10 am!) for brunch and some delicious cake. We also got work done and made an entire storyboard for the presentation. Our golden (or perhaps orange) idea of the day was a growing carrot representation for the carotenoid pathway. <br />
|tech=<br />
|author=Eric, Arun <br />
}}<br />
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<div id="week13entry" class="nb-week"><br />
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<h3 class="centered">Week 13</h3><br />
<h5 class="centered">(09/09 - 09/15)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 9th<br />
|text=We met with our design master Oat to discuss design considerations for the presentation. Oat will be making many designs, including a kill switch diagram, homologous recombination figure, our carotenoid image, a toolkit representation, and of course lots of mushrooms. <br />
|tech= <br />
|author=Eric<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 10th<br />
|text=Oat, Eric, Arun, and Hannah sat down to make figures for the presentation. Arun also chose his first color scheme: a baby blue flanked by a serene green. We got many designs done including the carotenoids, homologous regions, kill-switch, and the construct pieces.<br />
|tech=<br />
|author= Arun, Eric<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=September 13th<br />
|text= Managed to get the arduino to control the heating circuit! <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 15th<br />
|text=We once again got brunch and ate some school famous chocolate peanut butter cake. We agreed that Becker brunch has the best brunch. Then we sat down all day and put together the majority of the presentation including graphics and content. In addition, Hannah and Eric started writing the script for their part of the presentation. Arun will start writing his script as soon as characterization data starts coming in.<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
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<div id="week14entry" class="nb-week"><br />
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<h3 class="centered">Week 14</h3><br />
<h5 class="centered">(09/16 - 09/22)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 17th<br />
|text=We met for our first run through of our presentation. The script needs some work, but it’s coming along nicely. It seems that the only part lacking in the presentation is the characterization data and charts. They coming soon!<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 21st<br />
|text=We ran through our slides for the full team. We received a lot of good advice, and we still have a lot of work to do, but most of it will have to wait until after tuesday so we don’t fail our orgo exam! The main suggestion would be to change the theme of the presentation as the baby blue and serene green are too subtle to captivate attention. In addition, there are little glitches to the images.<br />
|tech=<br />
|author=Arun , Eric<br />
}}<br />
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<div id="week15entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 15</h3><br />
<h5 class="centered">(09/23 - 09/29)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 25th<br />
|text=We made a lot of changes to the presentation tonight, and it is an almost polished form now. Arun switched to his next color scheme: a deep Cornell red with baby blue banners. Eric has gotten his part of the script memorized. He will be speaking about the three components of our project. The fungal toolkit, and its implementation through the carotenoid pathway and antifungals expression. Hannah has also prepared her part on human practices. In addition she has prepared an analysis on the market trends towards bioplastics.<br />
|tech=<br />
|author=Arun, Eric<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=September 26th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. <br />
|tech=<br />
|author=Eric<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=pres<br />
|head=September 27th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. Arun also experimented with backgrounds of clouds, dirt, and toolkit to make the introduction slides very sharp. It look like we now have a solid design for our presentation.<br />
|tech=<br />
|author=Arun,Eric<br />
}}<br />
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<h5 class="centered">(09/30 - 10/06)</h5><br />
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<div id="week17entry" class="nb-week"><br />
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<h5 class="centered">(10/07 - 10/13)</h5><br />
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<div id="week18entry" class="nb-week"><br />
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<h5 class="centered">(10/14 - 10/20)</h5><br />
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<div id="week19entry" class="nb-week"><br />
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<h3 class="centered">Week 19</h3><br />
<h5 class="centered">(10/21 - 10/28)</h5><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/notebookTeam:Cornell/notebook2013-09-28T01:49:07Z<p>R.Lizarralde: </p>
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<!-- -------------------Notebook entries------------------- --><br />
<div id="weekAentry" class="nb-week" style="display: block"><br />
<div class="twelve columns"><br />
<h3 class="centered">January</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 5th<br />
|text=Applications for new Cornell iGEM members were due today-- we've got 57!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 16th<br />
|text=After an eternity of reading and a gigantic spreadsheet of evaluations, we're finally ready to start interviewing applicants-- 36 of them!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 25th<br />
|text=I'm really excited about having these new members on the team. There were many awesome interviews, but now we have to sit down and make hard decisions on who to take, as most of last year's members are graduating, so there's a limit to how many new members we can train. This is going to be a tough weekend.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=January 27th<br />
|text=After several hours of discussion, we decided on our 21 new members! Congratulations, Tim Abbott, Hannah Ajmani, Eric Appel, Ryan Ashley, Nupur Bhatt, Arun Chakravorty, Rebecca Chew, Sharlene Dong, Sara Gregg, Alex Han, Eric Holmes, Daniel Leach, Oat Luengvarinkul, Jeffrey Ly, Ritvik Sarkar, Mac Sennett, Prashant Sharma, Olya Spassibojko, Yoshiko Toyoda, and Kyle Wheeler!<br />
|author=Rafael<br />
}}<br />
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<div id="weekBentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">February</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 1st<br />
|text=We had our first full team meeting today with all the new members, introducing them to the iGEM competition, and did an icebreaker we call speed-taboodunit! For the next week, they have the assignment of analyzing a previous iGEM project (in groups), and explaining what the project was trying to accomplish, whether synthetic biology was a good approach for this, and reasons for their success or lack thereof.<br />
|tech=Speed-taboodunit is a mashup of speed-dating, taboo, and whodunit; the idea is that each person writes on a piece of paper something unusual about themselves, underlining the key words, then these pieces of paper are randomly redistributed. Everyone organizes in a speed-dating format, with two lines of people, and you have a minute to talk to them and try to find out if they're the person described on their piece of paper-- with the catch that they can't use any of the underlined words. People who successfully find their person leave the group, but otherwise after the minute everyone moves down the line. As for why we came up with this specific icebreaker, we wanted to hit two key requirements of icebreakers: they have to be fun, and they have to make specific accommodations for being shy or quiet. Because each person has the specific objective of finding their person, this helps them overcome the shyness, and as for the fun, everyone loves taboo!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 7th<br />
|text=Our first social event tonight went well! Hopefully with a few more of these we'll have some great team comradery and feel more comfortable around each other. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 9th<br />
|text=At our second team meeting we went over the past project analyses, as well as some biology review-- mostly molecular cloning. For the next week, we've assigned more past projects for them to outline in more detail.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 16th<br />
|text=Today we went over literature review, went over the past project outlines, and gave a two-week problem-oriented brainstorming and project outline assignment. Now that each member has looked at several past projects and analyzed them, we're moving into brainstorming for this year's project and practicing the development of project pitches.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=February 21th<br />
|text=We just started working on the wiki! Our first assignment: figuring out how wiki templates work. With templates the website would become much easier to edit, but for some reason HTML and wiki text don't seem to work very well together...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=February 23th<br />
|text=For the second week of this assignment, we compiled the brainstormed ideas and had members pick which ones they wanted to explore further. We also had a movie night planned, but it fell through :(<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=February 28th<br />
|text=Finally got templates to work! The trick was ending HTML right before wiki text, and starting HTML again right afterwards. Not very elegant, but it works.<br />
|author=Nupur<br />
}}<br />
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<div id="weekCentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">March</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 1st<br />
|text=We want to revamp the notebook page with a filter system that lets you sort by subteam. We set up the basic layout of the page, but writing the javascript is going to be another story.<br />
|tech=Check out our <html><a href=https://2012.igem.org/wiki/index.php?title=Team:Cornell/Sandbox/notebook&oldid=299789 target="_blank"> notebook</a></html> before anything worked!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 2nd<br />
|text=Groups over the previous week investigated phage therapy, piezoelectric bacteria, "sticky paper", logic gates / bistable switches, phytases, mycelia, and pressure-adaptive materials. We're keeping the results from those, but doing a second round of brainstorming over the next few weeks-- the idea is to allow the exploration of any ideas that arose while researching the previous round of ideas.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 5th<br />
|text=We just finished the javascript for filtering. Rafael also made cool subteam icons for the filter buttons.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=March 6th<br />
|text=I made a template for notebook entries and added styling to each entry.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 9th<br />
|text=We compiled our second round of ideas, including topics like methanotrophs, a CRISPR knockout system, and suppressor tRNA inducible expression systems. We also took our first team picture, and probably the only one that will have all of last year's and this year's members together!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=March 29rd<br />
|text=Spring break knocked out a couple of meetings, but it looks like some research was accomplished nonetheless! The phytase project idea is looking pretty promising-- it incorporates a lot of different disciplines, with stuff for our mechanical and electrical engineers to do, as well as modeling.<br />
|tech=A lot of iGEM teams we've looked at don't have much beyond the biology, so we really try hard to do interdisciplinary projects, as they provide a great opportunity for learning to work with different kinds of people.<br />
|author=Rafael<br />
}}<br />
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<div id="weekDentry" class="nb-week"><br />
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<h3 class="centered">April</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 6th<br />
|text=We've narrowed it down to four project ideas: smokestack reactors (capturing pollutants with bioreactors), incorporation of phytases into anaerobic digesters, mycelial materials, and using bacteria to address issues with artificial implantable kidneys.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 10th<br />
|text=We had a call today with Ecovative Design, a company that uses mushrooms to make a biodegradable styrofoam analogue-- our mycelial materials project idea would involve working with them to improve their product. It seems like our interests are aligned and that we should be able to work with them. Exciting! <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 13th<br />
|text=This meeting was dedicated to project pitches from each of the four ideas, followed by voting on which project we should pursue. Some of our graduate advisors also attended to provide a more seasoned perspective on the viability of each project. After some deliberation and spreadsheet magic, we decided on mycelial materials!<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 20th<br />
|text=Today we worked on team structure for the summer; Swati will be the overall team leader, with me and Mark coordinating wetlab, Olya taking meeting minutes, Mac running drylab, and Hannah will be managing human practices. Later on in the summer, we will also develop task groups. For the next week, we've also split into groups to research various aspects of the mycelial materials project.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=April 26th<br />
|text=We've compiled initial research on carotenoid pigments, transformation protocols, DNA that may be useful for a basidiomycete toolkit, and a few other topics. Over the next week we'll meet with several professors on campus who work with fungi to see what resources and advice they can provide.<br />
|author=Rafael<br />
}}<br />
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<div id="weekEentry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">May</h3><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=May 4th<br />
|text=We met with Professor Gillian Turgeon, who does fungal transformation with Cochliobolus heterostrophus, an ascomycotic corn pathogen. We can get a few of the genes we've been looking for from her, yay! We also met with Professor Teresa Pawlowska, who directed us towards some other fungi and resources for fungi, as well as provided some background on fungal biology. As for the rest of the semester, we're letting everyone off the hook-- we don't want anyone to fail their exams because of iGEM!<br />
|author=Rafael<br />
}}<br />
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<div id="week1entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered" >Week 1</h3><br />
<h5 class="centered">(06/17 - 06/23)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 17th<br />
|text=The drylab team met briefly on June 17 to begin discussing some ideas. We went over some of the growing methods for Ganoderma lucidum, brainstormed some non-packaging related ideas for a fungal biomaterial, and discussed how we could test some of the physical characteristics of the final product. We thought it might be a good idea to test the thermal conductivity if it were to be used as a drinking cup or something. We also determined that people probably do not want to drink out of a fungus cup. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 18th<br />
|text=We started bootcamp today, doing our first transformations with kit plate biobricks.<br />
|tech=We transformed parts containing carotenoid pathway genes ''crtE'', ''crtI'', ''crtB'', and ''crtY'' (BBa_K523022, BBa_K539119, BBa_K145001), as well as the T7 promoter (both in pIVEX2.3d as well as BBa_I712074), the T7 polymerase gene (BBa_K145001), and the transformation efficiency kit part (BBa_J04450).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 19th<br />
|text=The second day of bootcamp had more activity-- we PCR-amplified a variety of parts to biobrick them and get ready for making composite parts later.<br />
|tech=From the plasmid pUCATPH (pAh13G), we amplified the ''trpC'' promoter (P''trpC'', a constitutive fungal promoter) and the ''trpC'' terminator (T''trpC''). From pNG (pAg13H), we amplified ''nptII'', the geneticin (a plant/fungal antibiotic) resistance gene.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 20th<br />
|text=I finally started moving pages to the 2013 wiki! It's looking very empty right now, but Rafael made a gif of our logo with his mycodraw program to use as a homepage placeholder. We also decided to add a technical details section to each of our notebook entries so that we can separate the jargon from daily summaries.<br />
|tech=Check out the <html><a href=https://static.igem.org/mediawiki/2013/2/21/CornellStillGrowing.gif target="_blank">gif</a></html> if it's not on our homepage anymore. Originally Rafael converted his mycodraw program from java to javascript (which took a VERY long time), but the script only worked on some browsers :( We had to settle for the gif instead.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 20th<br />
|text=On our third day of bootcamp, we taught new members how to run gels, do minipreps, and run digests, continuing our preparation of biobrick parts from the previous two days.<br />
|tech=We miniprepped the ''crtEIB'' (BBa_K523022), ''crtY'' (BBa_K539119), and T7 (BBa_I712074) biobricks. The gels confirmed our PCRs for P''trpC'', T''trpC'', and ''nptII'', which we then digested to insert into pSB1C3.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 21st<br />
|text=The javascript for toggling technical details took a bit longer than expected, but we finished it up today.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 21st<br />
|text=Continuing with bootcamp, we column-purified the digests of PCR products and gel-purified the digested pSB1C3 backbone, dephosphorylated, and ligated, resulting in our first batch of biobricks or pre-biobrick parts.<br />
|tech=P''trpC'', T''trpC'', and ''nptII'' were column-purified, digested pSB1C3 backbone was gel-purified and dephosphorylated, and each was ligated into the pSB1C3 backbone.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 22nd<br />
|text=I added a flyout to notebook entries authors with a picture, short description, and link to their bio on the team page.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 22nd<br />
|text=Our penultimate day of bootcamp was a light one-- we only transformed cells with the constructs we'd made over the past four days. <br />
|tech=We transformed T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) constructs into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 23rd<br />
|text=We concluded bootcamp with colony PCRs to test whether each of the constructs was present. Unfortunately, the gels had no bands, so we grew overnight cultures and will screen the minipreps instead.<br />
|tech=We used Q5 PCRs with the standard biobrick primers VF2 and VR to attempt to confirm the presence of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R).<br />
|author=Rafael<br />
}}<br />
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<div id="week2entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 2</h3><br />
<h5 class="centered">(06/24 - 06/30)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=June 24th<br />
|text=The drylab team met to update each other on some of the preliminary research. The main focus of this meeting was the development of a growing protocol. Sara found a professor that we could contact in the material science department for help regarding the testing of material properties. Rebecca and Manny looked into what different aspects of the fungi we could model, such as growth, decay, etc. Ritvik brainstormed some more ideas regarding the potential uses of this material, and came up with the idea of using it as insulation. <br />
|author=Mac <br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 24th<br />
|text=I started working on a hypertexting script that will automatically recognize words on a page and add a flyout with a description. We can use this to recognize names for the author flyouts I implemented yesterday, as well as other keywords such as plasmid names and protocols.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 24th<br />
|text=We miniprepped our three constructs. Then we went back to sleep.<br />
|tech=Minipreps of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 25th<br />
|text=I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 25th<br />
|text=We did another PCR from the miniprepped plasmids to confirm the right sizes of inserts, then sent them in for sequencing.<br />
|tech=The Q5 PCRs of T''trpC'' (pC13Q), P''trpC'' (pC13P), and ''nptII'' (pCg13R) showed bands of the correct size, so sequencing with VF2 and VR was submitted to make sure nothing strange had happened.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=June 26th<br />
|text=The script is now working on all browsers except for Internet Explorer.<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 27th<br />
|text=We started cloning crt genes downsteam of the T7 promoter. Because the T7 promoter is too small to extract as a fragment, we're placing crt genes downstream, then transferring the promoter+gene fragments back to pSB1C3. Today we digested ''crtY'' and the T7 promoter plasmid, but the digestion of ''crtY'' failed. <br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). However, the gel showed only one band for each (what one would expect for pAK13D, but pC13B should have had two bands).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 28th<br />
|text=We redid the digestion of ''crtY'' and the T7 promoter plasmid from yesterday, apparently successfully this time. We also started liquid cultures of pBARGPE1, a plasmid we got from the Fungal Genetics Stock Center.<br />
|tech=pC13B (''crtY'') was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). The gel this time had two bands at the correct locations for pC13B, so we're hoping it's a success this time. We also started overnight LB cultures of pAb13T (pBARGPE1).<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 29th<br />
|text=Crossing our fingers, we gel extracted and ligated the ostensibly successful digests of ''crtY'' and the T7 promoter plasmid, and then transformed. We also digested ''crtI'' and ''crtB'', other carotenoid genes that we will also be putting under the T7 promoter, and miniprepped the pBARGPE1 that had been grown up last night.<br />
|tech=If successful, the ligation will form pAK13AD. pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI for insertion into pAK13D, and separated on a gel. pAb13T (pBARGPE1) was miniprepped and quantified by nanodrop. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=June 30th<br />
|text=Unfortunately, there were no colonies on the transformation plate for T7+''crtY'', so we redid the digestions for those. We also digested T''trpC'' and ''nptII'' for the eventual cloning of a P''trpC''+''nptII''+T''trpC'' construct (we have to put the parts together in reverse because ''nptII'' contains a PstI site, which we will be trying to remove as well). Gel extractions of digested ''crtI'' and ''crtB'' were also empty (insignificant concentrations), so these digestions were also redone, and all were run on a gel, which looked mostly okay.<br />
|tech=pC13K (''crtI'', BBa_K118003) and pC13M (''crtB'', BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (T''trpC'') with EcoRI and XbaI, and pCg13R (''nptII'') with EcoRI and SpeI, and all were separated on a gel; ''crtI'' looked faint, and pAK13D was smeared, but all fragments were cut out for purification tomorrow.<br />
|author=Rafael<br />
}}<br />
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<div id="week3entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 3</h3><br />
<h5 class="centered">(07/01 - 07/07)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 1st<br />
|text=We finished the hypertexting script and fixed some miscellaneous problems. I think we can call this a wrap. The notebook page is officially done!<br />
|tech=Now we just have to start filling out entries...<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 1st<br />
|text= Two of our team members participated in SILS Skills Night on June 20th.<br />
|tech= Price of one 12 oz Styrofoam cup: 4.09 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 1st<br />
|text=Yesterday's digestions were gel extracted, ligated, and transformed. Hopefully it worked this time!<br />
|tech=Digested pAK13D was dephosphorylated, then ''crtI'' , ''crtB'', and ''crtY'' were ligated with pAK13D to put them under the T7 promoter, hopefully producing pAK13AB, pAK13AC, and pAK13AD, respectively. pCg13R was also ligated with pC13Q, hopefully producing pCg13U. All four constructs were transformed into DH5α via electroporation.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 2nd<br />
|text=There were no transformants, again :(. On the other hand, we received our tube of ''Ganoderma lucidum'' from Dr. David Hibbett today! Once more with feeling, we redid the four digestion-ligations for ''crtI'', ''crtB'', ''crtY'', and ''nptII''. We also amplified ''hph'', the hygromycin resistance gene, from one of the plasmids we got from Dr. Turgeon's lab, as well as pSB1C3 backbone, and we will use Gibson assembly to put them together (as ''hph'' has both EcoRI and PstI sites)-- the gels looked good for both of these. Finally, we submitted pAb13T (pBARGPE1) for sequencing to determine the sequences of the ''Aspergillus nidulans'' ''gpdA'' promoter and the ''bar'' gene, which confers phosphonothricin resistance.<br />
|tech=''hph'' was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 3rd<br />
|text=All transformations failed yet again, and were repeated once more. Four tears shed for the lost. ''hph'' was Gibson-assembled, and we ran a Q5 PCR of the ''bar'' gene, under the assumption it matches other published ''bar'' sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on ''nptII'' (pCg13R) to remove its internal PstI site.<br />
|tech=:( ;_; T_T D: (tears for the transformations lost). The ''bar'' PCR and pC13F were digested with EcoRI and PstI, and ligated to hopefully form pCp13X. Site-directed mutagenesis was performed both with and without DpnI to digest template DNA. <add something about site-directed mutagenesis design; refer to primers? probably requires Swatinput><br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=July 5th<br />
|text=Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.<br />
|author=Nupur<br />
}}<br />
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<div id="week4entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 4</h3><br />
<h5 class="centered">(07/08 - 07/14)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 10th<br />
|text= Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 11th<br />
|text=After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop. <br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 11th<br />
|text=Digest screens were run to see if cloning had been successful,, and Gibson cloning was continued. <br />
|tech=Minipreps were made from the ''crtY'' plates, from which a digest screen was prepared using ''Not-I'' to see if the ligation worked. The digestions were run on a gel and some appear to have worked (X/P/S, X/P/A #2/3, and X/P/S/A #⅔ worked). Also, a glycerol stock of ''nptII BB'' was made and miniprepped, but the concentration was extremely low, so an overnight culture was made from the glycerol stock to be miniprepped tomorrow. Another transformation was done using the remaining miniprep of ''nptII BB'' from earlier. Cultures were made for ''Gibson #3'', and a colony PCR perfomed, then run on a gel. Non-GC fragments from Gibson #3 were too small, but GC #⅖ were good (1kb fragment), and #4 had a band at 1kb with another smaller fragment. Finally, ''nptII BB'' was digested with X/P then column purified, and ''PtrpC'' was digested with S/P then gel purified. <br />
<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 12th<br />
|text=Cloning with ''nptII BB'' and ''PtrpC'' was continued, as well as cloning with the Gibson Assembly method.<br />
|tech=Today ''nptII BB'' and ''PtrpC'' were quantified. Also, ''nptII BB'' transformants were miniprepped, and another glycerol stock of ''nptII BB'' was made. The DNA for Gibson Assembly was miniprepped, and GC colonies 2,4, and 5 from the Gibson #3 gel yesterday were prepared for sequencing. ''NptII BB'' was digested with the ''PtrpC'' digest from yesterday. More cultures of X/P/S #1/2/3, X/P/A #⅔, and X/P/S/A #⅔ were made. CYM medium was created, and ''PC13Z'' was transformed from a kit plate.<br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 13th<br />
|text=Today we worked with fungi! We applied different amounts of antibiotic to G. lucidum, to test the sensitivity of our strain of fungi. In addition, cloning of ''pCg13Y'' was nearly completed. <br />
|tech=''pCg13Y'' was desalted, transformed, then plated into E. coli. Antibiotic stocks ''G418'' and ''PPT'' were made (100x). G. lucidum cultures were inoculated with varying levels of antibiotic to test the strain's sensitivity for future selection purposes. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 14th<br />
|text=Our cloning of ''pCg13Y'' was found to be unsuccessful. ''pC13z'' and ''pAK13AD'' were prepped for cloning. <br />
|tech= Colony PCRs and cultures of ''pCg13Y'' were completed to determine the success of cloning ''nptII BB'' downstream of ''PtrpC''. Unfortunately, a gel revealed that the dna fragments appeared at 0.6kb instead of 1.4kb. ''pC13Z'' and ''pAK13AD'' were miniprepped, but had to be thrown out due to contamination of our EB. New cultures were made to be miniprepped again. A glycerol stock of ''PC13Z'' was made, too. <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 14th<br />
|text=This week, drylab split up so members could focus on their strengths. Rebecca and Sara began designing our feedback controller, with some much appreciated advice from the previous drylab team leader, Dan. Ritvik, Manny, and Mac began coming up with design considerations for the incubation chamber. We were most concerned with size; too small and it wouldn't be useful, too big and it would be difficult to control any variables. We eventually decided that a 1x1x2' chamber would probably be a good compromise. Rebecca, Sara, Ritvik, and Mac also attended machine shop training this week. In order to complete our training, we each had to construct a small aluminum lamp using a milling machine and a lathe. Fortunately, everyone emerged from the training session one lamp richer, and with a full set of fingers on each hand.<br />
|author=Mac <br />
}}<br />
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<div id="week5entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 5</h3><br />
<h5 class="centered">(07/15 - 07/21)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 15th<br />
|text=Rebecca looked into the components of the temperature feedback control; namely, the temperature sensor, the microcontroller and the heating element. Put in an order for the temperature sensor, microcontroller (an Arduino Fio) and Xbee ports for wireless communications.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 15th<br />
|text=Gibson ''hph'' was submitted for sequencing, and cloning of ''crtY'' and ''crtB'' was continued. <br />
|tech=Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. Gibson ''hph'' was submitted for sequencing, and ''pAK13AD''s and ''pC13Z''(GFP) were miniprepped. ''PC13Z'', ''PC13F'', ''PCP13X'', ''PC13M'', and ''PC13E'' were PCRed with primers 31 and 32, the standard ''pSB1C3'' primers. A gel was run to check the PCRs-all looked successful except ''pC13M'' and ''pC13E'' which had nonspecific bands around 1kb. A new technique was used in the following digests, which used a ''restriction cocktail'' of enzymes to cut the dna. ''PAK13AD'' was digested with E/S/SacI/ClaI so that it could be placed in ''PSB1C3'' using ''SpeI'' instead of ''PstI'' due to the small distance between the ''PstI'' and the ''SacI'' cut sites. ''PC13F'' was digested with E/S/KpnI so ''PAK13AD'' can be placed in it. ''PAK13D'' was digested with S/P so ''crtI''/''crtB'' can be placed in it. All the digests were then column purified rather than gel purified--a move which has improved our ligation efficiencies. Then the purifications were quantified. Cultures were made for ''PC13P'' and ''PC13K''. Finally, ''PAK13AD'' was dephosphorylated then ligated into ''PC13F'' (''PC13AH''). <br />
|author=Rafael<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 16th<br />
|text=One iGEM member spoke at a panel for pre-freshmen biology students interested in pursuing research positions in college (PSP).<br />
|tech= Price of one 12 oz Styrofoam cup: 4.95 cents<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 16th<br />
|text=<br />
|tech=Sequencing results came back positive for the Gibson cloning of hph, so a culture of gibson colony 5 was made. ''PC13AH'' was transformed and plated, and ''pC13K'' and ''pC13K'' were miniprepped. ''pCp13X'' (S/P), ''PC13F'' (S/P), ''PC13Z'' (S/P) were purified and digested along with ''PC13P'' (X/P) [also dephosphorylated], then all were column purified. Reran a PCR of ''PC13M'' and ''PC13E'', and one of ''PC13K'' with primers 31/32. All PCRs were cleaned, then run on a gel along with ''PBARGPE1'' and ''PBARGPE1'' which had been digested with NotI today. Results: ''PC13E'' had a 3kb fragment (good), ''PC13K'' had a .9kb fragment (should be 1.7-1.8kb), ''PC13M'' had a 1.2-1.5kb fragment (good), ''pBARGPE1'' had a 4kb fragment (super-coiled) and ''pBARGPE1'' with NotI had a 5-6kb fragment (has a NotI site-good). From these results it can be concluded that everything worked except ''PC13K''. Ligations of ''PAK13D'' to inserts ''PC13Z'', ''pCp13X'', and ''PC13F'' along with vector ''PC13P'' to inserts ''PC13Z'', ''pCp13X'', and ''PC13F'' were performed. Also, overnight digestions of ''PC13M'', ''PC13E'', and ''PC13K'' with X/P were set up. Transformation was attempted for ''PC13AI'' and ''PA13AJ'', but the cuvettes weren't cleaned properly so it failed. Finally, our dwindling supplies prompted the ordering of more Spe1 and membrane filters. <br />
|author=Rafael<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wet<br />
|head=July 18th<br />
|text=The restriction cocktail method of cloning worked! Cloning of pC13AH (''T7'' + ''crtY'') was shown to work using this method. We are also continuing our cloning of the ''crtI'' and ''crtB'' pathways. <br />
|tech=All 18 (yes, 18!) of the ''pC13AH'' colonies that had been screened by colony PCR yesterday were miniprepped. We digested the miniprepped colonies of pC13AH clones (T7 promoter with crtY in pSB1C3) with ''NotI'' to see if the restriction cocktail method of cloning worked. On the gel that was subsequently run, bands were expected at ~2.1 for pSB1C3 and ~1.2 for ''crtY''+''pT7''. The restriction cocktail method worked! The bands appeared at the correct lengths on the gel for ''pC13AH''. There was not enough miniprepped DNA for pC13AH to send for sequencing, so the colonies were cultured again for another miniprep. None of the ligations into pAK13D (pSB1AK8 + PT7) worked -- pC13Z (''GFP''), pC13M (''crtB''), and pCp13X (''bar'') all failed => try cloning only using restriction cocktail method, instead of attempting PCR with VF2 & VR. Digested pC13Z, pC13M, pCp13X, pC13K (''crtI'') with XbaI and PstI in addition to ApaLI and SacI for later ligation with pAK13. pAK13D had been previously digested with SpeI and PstI, column purified, and dephosphorylated for insert ligation. More pAK13D was grown to acquire more DNA to be miniprepped tomorrow. Nupur performed colony PCR and made cultures of transformants from yesterday. All transformations appeared to have very low efficiency--perhaps an issue with heat shock stocks? Transformations of ''AI-AL'' (mRFP cds and lox site parts) all yielded colonies, though at really low efficiencies compared to most transformations off of kit plates. These were screened by colony PCR to verify. Ligations into pC13P all yielded colonies, but at very low efficiencies. These were pC13E (T7 pol), pCp13X (bar), and pC13Z. All were screened by colony PCR as well. Taq was used to perform 5 colony PCRs, then all colony PCRs were run on a gel. Also, pCg13s (nptIIBB)was digested with XbaI, PstI, ApaLI, and SacI for later ligation to pC13P, which had been previously digested with SpeI and PstI, column purified, and dephosphorylated. Overnight ligations of pC13Z, pC13M, pCp13X, pC13K, pCg13S with vector pAK13D were set up, along with overnight ligations of pC13K and pCg13S in vector pC13P. <br />
|author=Danielle<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 21st<br />
|text=This week, the fabrication group focused on what materials to construct our chamber from. At first we considered a single wall design, but this was eventually scrapped, as we were unable to find any material that would provide sufficient insulation at a reasonable thickness (and cost). After putting our heads together, we decided on a double wall with an insulating layer in between them. Mac began drawing up the chamber in Solidworks. <br />
|author=Mac <br />
}}<br />
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<div id="week6entry" class="nb-week""><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 6</h3><br />
<h5 class="centered">(07/22 - 07/28)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 23rd<br />
|text=The parts have arrived and Rebecca configured them. Met up with Paras who was on last year's team to get a detailed explanation of how their project had worked as well.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=July 23rd<br />
|text=Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 28th<br />
|text=This week, the fabrication group finalized the incubator design and ordered the construction materials! We decided to construct the outer and inner layers out of HDPE plastic in the rather lab appropriate color of white. For our insulation, we decided to use high density polystyrene. After comparing the K-factors for various types of insulation, we determined that this material gave us the most bang for our buck, so to speak. <br />
|author=Mac <br />
}}<br />
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<div id="week7entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 7</h3><br />
<h5 class="centered">(07/29 - 08/04)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=July 31st<br />
|text=Sara and Rebecca met up with our advisor Dr Bruce Land and he provided lots of helpful advice with regard to the design considerations (where the heater should be in our incubation chamber, convection currents, type of heating plate to use). He also suggested that we use a proportional-integral-derivative controller (PID controller) to regulate the heating.<br />
|author=Rebecca<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 1st<br />
|text= Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi. <br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 2nd<br />
|text= Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.<br />
|author=Hannah<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 4th<br />
|text=Mac went to visit Ecovative Design with Swati, Rafael, Alex, and Hannah. We were able to meet with Gavin McIntyre, one of the founders of the company, who was kind enough to give us a tour of their facilities. He also gave our wetlab members some pointers for our own project. In other news, we are still waiting for our materials to arrive. <br />
|author=Mac <br />
}} <br />
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<div id="week8entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 8</h3><br />
<h5 class="centered">(08/05 - 08/11)</h5><br />
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{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 5th<br />
|text= Eric Holmes and Arun Chakravorty presented for a group of scientific researchers (SILS)<br />
|author=Hannah<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=wiki<br />
|head=August 7th<br />
|text=We helped [[Team:Ciencias-UNAM|Ciencias-UNAM]] get set to transfer their HTML website to the wiki. Hopefully we'll get up a tutorial for what to look out for on here soon!<br />
|author=Nupur<br />
}}<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 7th<br />
|text=The sensor has been connected! <br />
|author=Rebecca<br />
}}<br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=hprac<br />
|head=August 10th<br />
|text= Five team members presented at the Ithaca Sustainability Center for citizens of Tompkins County.<br />
|author=Hannah<br />
}}<br />
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<div id="week9entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 9</h3><br />
<h5 class="centered">(08/12 - 08/18)</h5><br />
</html><br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 15th<br />
|text=Rebecca looked more into the details of the pulse-width modulation and PID controller so that the heater can be turned on for certain periods of time for more even heating. <br />
|author=Rebecca<br />
}}<br />
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{{:Team:Cornell/templates/nbentry<br />
|type=anim<br />
|head=August 18<br />
|text=Today, the dastardly trio of Arun, Hannah, and Eric were chosen to present at the regional iGEM competition!<br />
|tech=<br />
|author=Swati<br />
}}<br />
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<div id="week10entry" class="nb-week"><br />
<div class="twelve columns"><br />
<h3 class="centered">Week 10</h3><br />
<h5 class="centered">(08/19 - 08/25)</h5><br />
</html><br />
<br />
{{:Team:Cornell/templates/nbentry<br />
|type=dry<br />
|head=August 25th<br />
|text=Christine's back! She started looking at the components for a humidity sensor and placed an order for a mistmaker which would be able to control humidity levels. In the meantime, Sara studied the details of the heating circuit which utilizes a transistor to amplify signals and switches it such that the resistor could be used as the heating element. <br />
|author=Rebecca<br />
}}<br />
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</div><br />
<div id="week11entry" class="nb-week"><br />
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|head=August 26th<br />
|text=Our materials are here and everyone in the fabrication group has returned to Ithaca! This week, Manny and Mac focused their efforts on getting the plastic cut to the proper dimensions. Unfortunately, this proved more to be more difficult than anyone could have imagined. After transporting a rather hefty 48"x48" piece of plastic across campus from Weill Hall to the Emerson Lab machine shop, we were told that we would have to take it elsewhere to have it cut (plastic is bad for blades). Disappointed, but never defeated, we decided to try our luck with another machine shop. Also, carrying plastic around campus isn't much fun, so we held it on the roof of a car instead (not recommended). Fortunately, no traffic violations were received, and the Clark hall machine shop was more than happy to cut up our plastic for us. <br />
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|text=Lydia's back too! Discussed what we have been doing for the past few weeks and immediate goals. <br />
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|text=Christine, Rebecca and Sara met up with Dr. Land again and we learnt more about setting up PID controls. Went to the lab next where Christine and Sara set up the heating element circuit, Rebecca soldered pins onto the arduino and Lydia drew up the circuit design.<br />
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|text= We met at Flora Rose house to decide on an outline of the presentation and also decide a timeline.We met for the first time to discuss a preliminary outline for the presentation. Hannah will be handling all the human practices elements, Eric will be explaining relevant biological background and strategies, and Arun will be presenting all of our characterization data (hopefully!). We made a detailed structure which will include an introduction about styrofoam pollution and ecovative. Then we will move on to a brief overview of our wetlab work. It was clear that the wetlab portion should be divided into 3 main components. The components would be fungal toolkit, carotenoid pathway, and antifungals. Finally, we will end with results, human practices, and acknowledgments to all our beautiful advisors and sponsors.<br />
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|text= Five team members went to the Ithaca Sciencenter and taught a group of 18 children and 17 parents about the power of DNA.<br />
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|text= After researching we found out that the powerpoint presentation should be centered around design and images rather than content. We decided to meet tomorrow to discuss possible design layouts and schemes. We looked at many examples of presentations and considering our project, we think we should have a very modern and green theme. Arun will explore with this later on.<br />
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|text=We met super early in the morning (10 am!) for brunch and some delicious cake. We also got work done and made an entire storyboard for the presentation. Our golden (or perhaps orange) idea of the day was a growing carrot representation for the carotenoid pathway. <br />
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|text=We met with our design master Oat to discuss design considerations for the presentation. Oat will be making many designs, including a kill switch diagram, homologous recombination figure, our carotenoid image, a toolkit representation, and of course lots of mushrooms. <br />
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|text=Oat, Eric, Arun, and Hannah sat down to make figures for the presentation. Arun also chose his first color scheme: a baby blue flanked by a serene green. We got many designs done including the carotenoids, homologous regions, kill-switch, and the construct pieces.<br />
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|text= Managed to get the arduino to control the heating circuit! <br />
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|text=We once again got brunch and ate some school famous chocolate peanut butter cake. We agreed that Becker brunch has the best brunch. Then we sat down all day and put together the majority of the presentation including graphics and content. In addition, Hannah and Eric started writing the script for their part of the presentation. Arun will start writing his script as soon as characterization data starts coming in.<br />
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|text=We met for our first run through of our presentation. The script needs some work, but it’s coming along nicely. It seems that the only part lacking in the presentation is the characterization data and charts. They coming soon!<br />
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|text=We ran through our slides for the full team. We received a lot of good advice, and we still have a lot of work to do, but most of it will have to wait until after tuesday so we don’t fail our orgo exam! The main suggestion would be to change the theme of the presentation as the baby blue and serene green are too subtle to captivate attention. In addition, there are little glitches to the images.<br />
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|head=September 25th<br />
|text=We made a lot of changes to the presentation tonight, and it is an almost polished form now. Arun switched to his next color scheme: a deep Cornell red with baby blue banners. Eric has gotten his part of the script memorized. He will be speaking about the three components of our project. The fungal toolkit, and its implementation through the carotenoid pathway and antifungals expression. Hannah has also prepared her part on human practices. In addition she has prepared an analysis on the market trends towards bioplastics.<br />
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|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. <br />
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|author=Eric<br />
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|head=September 27th<br />
|text=Today we met again with Oat to make some changes to our images. Arun moved onto his next color scheme: the same deep maroon with elephant grey banners. In addition, the data, and ending slides will follow a baby blue and off white color scheme. We also made a few of our slides (our styrofoam map, standoff figure, and toolkit image) fancier and more attractive. Arun also experimented with backgrounds of clouds, dirt, and toolkit to make the introduction slides very sharp. It look like we now have a solid design for our presentation.<br />
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</html></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/project/future/pharmaceuticalsTeam:Cornell/project/future/pharmaceuticals2013-09-28T01:45:07Z<p>R.Lizarralde: fixed unclosed h3</p>
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<h2 class="centered">Pharmaceuticals</h2><br />
Many clinically used drugs are derived from fungi. A few notable examples include the antibiotic penicillin, immunosuppressant cyclesporin A and the statins lovastatin and squalestatin, which act to reduce and remove low density lipoproteins from blood vessels and lower chance of heart attack, stroke and diabetes. Fungi are extremely useful in biotechnology efforts and pharmaceutical research because they are able to utilize many unique metabolic pathways to produce a wide array of bioactive compounds capable of producing many positive pharmacological effects [1]. The biocompounds synthesized our organism, <i>Ganoderma lucidum</i>, are capable of regulating the immune system, inhibiting platelet aggregation, reducing blood pressure, and lowering levels of cholesterol and sugars. In addition, <i>Ganoderma lucidum</i> has the potential to be used against the Human Immuno Virus (HIV), exhibits anti-tumor activity and can propagate anti-diabetic effects. [2] <br />
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<i>Ganoderma lucidum</i> can also exhibit many immuno-modulating effects by activating T-cells and macrophages, which act as the body's first line of defense by releasing cytokines, nitric oxides (NO) and other mediators. These cytokines and NOs are important chemical messengers for inducing biological responses. In the case of Human Immuno Virus (HIV) and Acquired Immuno - Deficiency Syndrome (AIDS), <i>Ganoderma lucidum</i> produces many natural products that can serve as anti-HIV agents. Triterpenoids such as lucidenic acid O and lucidenic lactone inhibit HIV-1 Reverse Transcriptase, an enzyme encoded by HIV to aid in the reproduction of the virus and propagate mutations to develop polyproteins to create the main protein components of an infectious HIV virion [3, 4]<br />
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A <i>G. lucidum</i> polysaccharide GLB7 also decreases the production of oxygen free radicals and exerts an anti-mutagenic and anti-tumor effect. One major mechanisms of anti-tumor action includes polysaccharide -mediated potentiation of immune function, which is mostly due to beta-D glucans, which bind to leucocyte surfaces and serum-specific proteins and increase production of cytokines, interleukins and interferons, nitric oxides and antibodies [5-13]. Other mechanisms include suppressing the growth of leukemia cells and inducing differentiation into mature erythrocytic cells [14], inhibition of DNA polymerase and post-translational modification of oncoproteins [15-17]. <br />
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<i>Ganoderma lucidum</i>’s anti-diabetic effects include hypoglycemic and hypolipidemic activities and inhibition of increased blood glucose without raising blood insulin levels [18]. This effect is supported by clinical tests in patients with confirmed type II diabetes mellitus who experienced lowered blood glucose concentrations after treatment with Ganopoly (polysaccharide fractions extracted from <i>G. lucidum</i>) [19]. Other effects of the fungi vary from anti-allergic to estrogenic and hepatoprotective; a thorough review of <i>Ganoderma lucidum</i>'s possible pharmacological effects is available [2]. <br />
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The various biocompounds synthesized from this strain of fungi poses many possibilities for pharmacological development. However, there are also many challenges present in this endeavor. Pharmaceutical exploitation is still very limited due to the limited quantity of <i>Ganoderma lucidum</i> found in nature. Polysaccharides, ganoderic acids and other metabolites have mostly been extracted from solid state cultivated fruit bodies but methods of extraction from liquid cultivated mycelium still need to be thoroughly investigated [2].<br />
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<h3>References</h3><br />
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[1] Wainwright M. (1995) An Introduction to Fungal Biotechnology. Wiley, Chichester. <br /><br /><br />
[2] Sanodiya, B.S.; Thakur, G.S.; Baghel, R.K.;Prasad, G.B.K.S.;Bisen, P.S. (2009) <i>Ganoderma lucidum</i>: A Potent Pharmacological Macrofungus. <i>Current Pharmaceutical Biotechnology</i>, <b>10</b>, 717-742.<br /><br /><br />
[3] Kartikeyan, S.; Bharmal, R.N.; Tiwari, R.P. and Bisen, P.S. (2007) <i>HIV and AIDS: Basic Elements and Priorities</i>, Springer Verlag: Dordrecht, The Netherlands.<br /><br /> <br />
[4] Menendez-Arias, L. (2002) Targeting HIV: antiretroviral therapy and development of drug resistance. <i>Trends Pharmacol. Sci</i>., <b>23</b>, 381-388.<br /><br /> <br />
[5] Miyazaki, T. and Nishijima, M. (1981) Studies on fungal polysaccharides. XXVII. Structural examination of a water-soluble, anti- tumor polysaccharide of <i>Ganoderma lucidum. Chem. Pharm. Bull</i>., <b>29</b>, 3611-3616.<br /><br /> <br />
[6] Usui, T.; Iwasaki, Y.; Hayashi, K.; Mizuno, T.; Tanaki, M.; Shinkai, K. and Arakawa, M. (1981) Antitumor activity of water- soluble beta-D-glucan elaborated by <i>Ganoderma applanatum. Ag- ric. Biol. Chem</i>., <b>45</b>, 323-326. <br /><br /><br />
[7] Usui, T.; Iwasaki, Y.; Mizuno, T.; Tanaki, M.; Shinkai, K. and Arakawa, M. (1983) Isolation and characterization of antitumor ac- tive beta-D-glucans from the fruit bodies of <i>Ganoderma applanatum. Carbohydr. Res.</i>, <b>115</b>, 273-280. <br /><br /><br />
[8] Sone, Y.; Okuda, R.; Wada, N.; Kishida, E. and Misaki, A. (1985) Structural and anti-tumor activities of polysaccharides isolated from fruiting body and the growing culture of mycelium of <i>Gano- derma lucidum. Agric. Biol. Chem<.i>., <b>49</b>, 2641-2653. <br /><br /><br />
[9] Kishida, E.; Okuda, R.; Sone, Y. and Misaki, A. (1988) Fractiona- tion structures and antitumor activities of the polysaccharides of Reishi, the fruiting body of <i>Ganoderma lucidum. Osaka-Shiritsu Daigaku Seikatsukagakubu Kiyo</i>, <b>35</b>, 1-10. <br /><br /><br />
[10] Konopski, Z.; Smedsrod, B.; Seljelid, R. and Eskeland, T. (1994) A novel immunomodulator soluble aminated ß-1,3-D-glucan: binding characteristics to mouse peritoneal macrophages. <i>Biochem. Biophys. Acta</i>, <b>1221</b>, 61-65. <br /><br /><br />
[11]Mueller, A.; Raptis, J.; Rice, P.J.; Kalbfleisch, J.H.; Stout, R.D.; Ensley, H.E.; Browder, W. and Williams, D.L. (2000) The influ- ence of glucan polymer structure and solution conformation on binding to (13)-beta-D-glucan receptors in a human monocyte- like cell line. <i>Glycobiology</i>, <b>10</b>, 339-346. <br /><br /><br />
[12] Battle, J.; Ha, T.Z.; Li, C.F.; Dellabeffa, V.; Rice, P.; Kalbfleisch, J.; Browder, W. and Williams, D. (1998) Ligand binding to the (13)-beta-D-glucan recpetor stimulates NF-kappa B activation, but not apoptosis in U937 cells. <i>Biochem. Biophys. Res. Commun.</i>, <b>249</b>, 499-504. <br /><br /><br />
[13] Muller, A.; Rice, P.J.; Ensley, H.; Coogan, P.S.; Kalbfleisch, J.H.; Kelley, J.L.; Love, E.J.; Portera, C.A.; Ha, T.Z.; Browder, I.W. and Williams, D.L. (1996) Receptor binding and internalization of a water-soluble (13)-beta-D-glucan biologic response modifier in two monocyte macrophage cell lines. <i>J. Immunol.</i>, <b>156</b>, 3418-3425. <br /><br /><br />
[14] Zhong, L.; Jiang, D.Z. and Wang, Q.R. (1999) Effects of <i>Ganoderma lucidum</i> (Leyss ex Fr) Karst compound on the proliferation and differentiation of K562 leukemic cells. <i>J. Hunan Med. Univ</i>., ,<b>24</b>, 521-524. <br /><br /><br />
[15]Mizushina, Y.; Hanashima, L.; Yamaguchi, T.; Takemura, M.; Sugawara, F.; Saneyoshi, M.; Matsukage, A.; Yoshida, S. and Sakaguchi, K. (1998) A mushroom fruiting body-inducing sub- stance inhibits activities of replicative DNA polymerases. <i>Biochem. Biophys. Res. Commun.</i>, <b>249</b>, 17-22.<br /><br /><br />
[16]Mizushina, Y.; Watanabe, I.; Togashi, H.; Hanashima, L.; Takemura, M.; Ohta, K.; Sugawara, F.; Koshino, H.; Esumi, Y.; Uzawa, J.; Matsukage, A.; Yoshida, S. and Sakaguchi, K. (1998) An ergos- terol peroxide, a natural product that selectively enhances the in- hibitory effect of linoleic acid on DNA polymerase beta. <i>Biol. Pharm. Bull</i>., <b>21</b>, 444-448. <br /><br /><br />
[17]Lee, S.; Park, S.; Oh, J.W. and Yang, C.H. (1998) Natural inhibi- tors for protein prenyltransferase. <i>Planta Medica</i>, <b>64</b>, 303-308. <br /><br /><br />
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[18]Mohammed, A.; Adelaiye, A.B.; Abubakar, M.S. and Abdurahman, E.M. (2007) Effects of aqueous extract of <i>Ganoderma lucidum</i> on blood glucose levels of normoglycemic and alloxaninduced dia- betic wistar rats. <i>J. Med. Plants Res.</i>, <b>1</b>(2), 34-37. <br /><br /><br />
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[19]Gao, Y.; Lan, J.; Dai, X.; Ye, J. and Zhou, S.H. (2004) A phase I/II study of Ling Zhi mushroom <i>Ganoderma lucidum</i>. (W. Curt.: Fr.) Lloyd (Aphyllophoromycetideae) extract in patients with type II diabetes mellitus. <i>Int. J. Med. Mushrooms</i>, <b>6</b> (1), 33-39. <br /><br /><br />
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<h3 class="centered" style="margin:0px"> We have developed a toolkit to allow various industries to use fungi to develop sustainable technologies.</h3><br />
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<h4 class="centered">Background</h4><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background/fungal_toolkit">Fungal Toolkit</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background/fungal_genetic_engineering">Fungal Genetic Engineering</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background/ecovative">Ecovative</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/background/impact">Environmental Impact</a><br />
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<h4 class="centered">Wetlab</h4><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/chassis">Chassis</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit">Fungal Toolkit</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/protoplasting">Protoplasting</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/regulatory_elements">Regulatory Elements</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/selectable_markers">Selectable Markers</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/characterization">Characterization</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/homologous_constructs">Homologous Constructs</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/biosafety">Biosafety</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/carotenoids">Carotenoids</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/fungal_toolkit/antifungals">Antifungals</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/biobricks">BioBricks</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/wetlab/future_work">Future Work</a><br />
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<h4 class="centered">Drylab</h4><br />
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<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab">Overview</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab/design_considerations">Design Considerations</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab/components">Components</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab/components/fabrication">Fabrication</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/drylab/components/electronics">Electronics</a><br />
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</li><br />
<li><br />
<a href="https://2013.igem.org/Team:Cornell/project/drylab/modeling">Modeling</a><br />
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<a href="https://2013.igem.org/Team:Cornell/project/hprac/economics">Economic Analysis</a><br />
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<li><br />
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</li><br />
<li><br />
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</li><br />
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</html></div>R.Lizarraldehttp://2013.igem.org/File:CornellMicroryza940x329.pngFile:CornellMicroryza940x329.png2013-09-28T01:42:26Z<p>R.Lizarralde: </p>
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<div></div>R.Lizarraldehttp://2013.igem.org/Team:CornellTeam:Cornell2013-09-28T01:32:22Z<p>R.Lizarralde: </p>
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</html></div>R.Lizarraldehttp://2013.igem.org/File:CornellNovelty.jpgFile:CornellNovelty.jpg2013-09-28T01:31:56Z<p>R.Lizarralde: </p>
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<div></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/team/biosTeam:Cornell/team/bios2013-09-28T01:27:14Z<p>R.Lizarralde: </p>
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<body><br />
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<li><br />
<h6>Team</h6><br />
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<h2 class="centered">Team Members</h2><br />
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<div class="row" id="abbot"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/6/62/Abbott_tim.jpg" /><br />
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<h3>Tim Abbott</h3><br />
Tim knows who you are. And he knows what you want. If you're looking to unravel the secrets of the antisense RNA strand, he will falsely say that he doesn't know, but what he does know is a particular set of skills. Skills acquired over a long career that make him very dangerous to cells, so that they cannot hide their classified intel from him. Recruited by the the iGEM intercellular coalition (in a microcosm far away) to prevent worldwide biological warfare and Malthusian catastrophe, secret agent TR Abbott is armed with the cunning and intellect that parallels the legendary 007 himself. In the shadowy dark of night, he scales the highest chitin walls, dodges laser detection beams and disables all nucleus security defenses to deftly reprogram all cell circuitry before you can type <Hacksaur ftw n00b.> In the stark light of day, the dapper gentleman woos women with his brilliant smile and golden locks, bakes Thirty-Minute Brownies in twenty minutes and makes dirty dishes disappear. He struts not walks, he dodges and frolics, and all his bills are paid. He sleeps once a week, and when he does, it is in front of his computer. With his eyes open. He's Tim. Timothy Robert Abbott. And he is always watching. He will look for you, he will find you, and he will ki-- <br />
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<div class="row" id="ajmani"><br />
<div class="nine columns"><br />
<h3>Hannah Ajmani</h3><br />
Affectionately nicknamed Hanana, she is best known as the Caucasian whose actually 100% Indian, the CTB addict, and the girl who managed to burn a microwavable brownie.<br />
She hails from Jersey, produces movies in Cali (watch Sharknado, it's so bad... it's good). And guys, she can rap. Seriously, isn't this girl cool?! She's the last person you'd expect on Cornell iGEM, but the first person to lecture you on electroporation and/or inoculation if you ask. I think she has a secret liking for biology ...don't ask her though, she'll only deny it. When she isn't pretending to dislike biology, she puts on the most kick-ass outreach programs in iGEM history (...no offense Swati).<br />
Hannah is an amazing person be around. Her giggles sparkle, her smile is contagious, you can't help but love her. If you ever feel a little down, read one of her emails (seriously guys, read her emails) and you'll be sure to see her motto... ""Get excited people!""<br />
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<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/1/18/Ajmani_hannah.jpg" /><br />
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<div class="row" id="appel"><br />
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<img src="https://static.igem.org/mediawiki/2013/9/94/Appel_eric.jpg" /><br />
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<h3>Eric Appel</h3><br />
Eric is the enigmatic mastermind of our team. His quiet, devious intrigues behind the scenes ensure the team’s success at every endeavor. The moment he begins to fade from thought, he returns with divine vigor and grants the team the strength and resolve to confront the treacherous perils of necrotizing fungi and ethidium bromide. You can rest easy around Eric, confident that he has 23 years of experience, the master plan to the universe and your current home address. Don’t let his bright, jovial countenance fool you; Eric is the true power that will bring about our iGEM dominance.<br />
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<div class="row" id="ashley"><br />
<div class="nine columns"><br />
<h3>Ryan Ashley</h3><br />
Ryan James Ashley, at first glance, seems to be a quiet, unassuming fellow. Often referred to as “the only one who’s actually working,” Ryan is known for his unwavering focus, matched only by his superhuman productivity. Little did we know, however, that Ryan had all of us fooled. Ryan is impervious to pain, insensitive to temperature fluctuations, and unlimited in his capacity for retaining knowledge. He had mastered every martial art form in the universe by his twelfth birthday. Since then, he has looked to dominating the dance floor. Having already prevailed over the more exotic varieties, he can now be found salsa-ing his way into onlookers’ hearts at every turn. In his spare time, he has been known to traipse about the Norwegian countryside with an army of mountain goats.<br />
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<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ea/Ashley_ryan.jpg" /><br />
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<div class="row" id="bhatt"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cf/Bhatt_nupur.jpg" /><br />
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<div class="nine columns"><br />
<h3>Nupur Bhatt</h3><br />
Standing in the purple light of the cave, something seems amiss. The beautifully morose bass music that drew you here, almost against your will, has faded to a single, wavering note in the darkness. A figure is approaching, a calm grin just visible atop her slight frame. Suddenly, you are afraid. You raise your longsword just as your vision succumbs to a fiery halo; then, you open your eyes groggily. A scene from Skyrim flickers from the screen on Nupur's Thinkpad; yet, during your nap, ninety-six minipreps were completed, the lab reorganized, and the wiki redesigned and updated. You grab a box of pipette tips to continue your mastermix, only to find an intricate design in the negative space. A calm grin rests on Nupur's face.<br />
</div><br />
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<div class="row" id="chakravorty"><br />
<div class="nine columns"><br />
<h3>Arun Chakravorty</h3><br />
The ladies of iGEM once tried to expel Arun from the team for his distracting good looks. He simply <div class="author-hover"><a href="https://static.igem.org/mediawiki/2013/8/82/CornellArunWink.gif">winked at them</a></div><div class="author-hover-pic"><div class="author-desc" style="height:180px;width:180px"><img src="https://static.igem.org/mediawiki/2013/8/82/CornellArunWink.gif" style="width:166px;height:166px"><div></div></div></div> and they changed their minds. Armed with his sparkling smile, baby soft cheeks, and boyish charm, Arun will melt your heart (and your agar plates). But don’t be fooled. This dastardly devil will always surprise you. One moment he’ll be asleep, the next he’ll be chair jousting. One minute he’ll be serenading you with sweet sounds from his scrumptious, supple lips; the next minute he’ll be rapping B.I.G. One day he’ll be clean shaven, ten years later, he’ll maybe have a five o’clock shadow. Despite these bipolar tendencies, two things remain constant in Arun’s life: he has a flair for dramatic pipetting and the ladies love him. But I beg you ladies, calm yourselves and heed my last warning: he will take your grandpa’s style; he will take your grandpa’s style. If you ever need it back, he will still be in Ithaca. But actually though. <br />
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<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c6/Chakravorty_arun.jpg" /><br />
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<div class="row" id="chen"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/4/49/Chen_jonlin.jpg" /><br />
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<h3>Jonlin Chen</h3><br />
The Cornell iGEM team has a secret weapon. Her name: J-Chen. As a locally-grown, ‘hood-dwelling laboratory superhero, J-Chen skillfully navigates through the ins and outs of Ithaca in her Ganodermobile. Though seemingly unintimidating at first glance, beware: she can whip out her legendary ice cream maker at any given moment, and coat you in a lemony flavor of happiness so fast that you’ll forget what you were cloning. Not only is she powerful herself, her faithful minions, Solutions I, II, and III, are always within arms’ reach. Don’t dare question J-Chen’s seniority, either, or else she’ll hold your precious 97 cultures hostage, until you provide a box of those sacred yellow tips as ransom. And if there’s ever miniprepping to be done, don’t worry about it – J-Chen has already gotten started.<br />
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<div class="row" id="chew"><br />
<div class="nine columns"><br />
<h3>Rebecca Chew</h3><br />
The one and only Rebecca Chew, </br><br />
From wetlab to drylab, there's nothing this chemical engineer can't do. </br><br />
Her efforts to build the team's fungal growth chamber are so great, </br><br />
That our fungi will soon grow taller than the Empire State. </br><br />
Outside of lab, Rebecca enjoys chocolate desserts and midnight strolls through the park, </br><br />
And secretly wishes she was either Rihanna or a great white shark. </br> <br />
So here are a few last words for Rebecca Chew, </br> <br />
You are amazing, where would we be without you?<br />
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<h3>Sharlene Dong</h3><br />
Ding-Dong! Who’s there? Bing-Dong! Hello? Ring-Dong! Who goes there? Ring-a-ling Dong! Surely, someone’s there? Lene-Dong! Sharley, please introduce yourself. Sharlene Dong!<br />
What could be said about Sharlene Dong? Apart from her jovial laugh and smile, she can salsa dance ‘till the cows come home, or rather, until 1am when the Big Red Barn closes. Regardless, she is an undergraduate Pre-Med Chemical Engineer that also happens to be a member of iGEM – makes one wonder how she has enough time for anything! Despite her hectic schedule, she still manages to conduct research on the calcium channels in the heart and enjoy the companionship of her iGEM teammates.<br />
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</div><br />
<div class="row" id="gregg"><br />
<div class="nine columns"><br />
<h3>Sara Gregg</h3><br />
Sara is a fellow Clevelander who can aim for any goal like Hawkeye can hit all of his targets. One word, LEGENDARY. She has the endurance of a turtle but the speed of the rabbit to run for miles everywhere, beating all of us to Moosewood. Even more, she can consume a common frozen internationally famed desert faster than it takes for a sweetened, fluffy dairy solid to phase change into a pool of sticky coco-liquid. Looking past this girl's legendary fame, she's got a brightening smile which will command innocent looking mushrooms to transform into dangerous beautifully-colorful homes for the Smurfs. Watch out for this one...<br />
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<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e4/Gregg_sara.jpg" /><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4e/Han_alex.jpg" /><br />
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<h3>Alex Han</h3><br />
Alex Han is CUGEM’s secret weapon. He can whip up anything from multiple Asiago cheese dishes to fungi models with his mastery of statistics! He has travelled all over the world, from studying abroad in Denmark (learning the language and even more wonderful recipes!) to representing Cornell in Turkey! Also a jokester, he does not hesitate to <del>creep on</del> be friendly to other members to make their days that much better! :)<br />
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<div class="row" id="holmes"><br />
<div class="nine columns"><br />
<h3>Eric Holmes</h3><br />
Unlike most humans, Eric T.S. Holmes was born from an orange flower on the tallest branch of the great sequoia in Oregon. At the moment of his birth, lightning tore across the sky only to strike his fragile body. He then grew wings, slew a few dragons, and then decided he wanted to be an iGEM member. His full name, Eric Taylor Swift Holmes, warned his interviewers that he was trouble when he walked in. And yet, the iGEM gods smiled upon him in his most desperate hour, knowing that the female singer (who had been named in his honor) would be devastated by the grief of her beloved. A master of all knots and traps, he has since conquered every terrain in the universe, including the fiery depths of the autoclave room. He now looks to the vast waters of Cayuga Lake in search of a legendary fish, one that has only been spotted once, by him of course. He captured only a picture but now thirsts for the creature's carcass, to avenge his lost left wing. In obsession, he surrounds himself with images of the deadly beast, <i>Arunicus chakravorticum</i>. He hopes that with his newly honed abilities from being an iGEM wet lab minion, he will be able to take down the creature. <br />
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<img src="https://static.igem.org/mediawiki/2013/9/94/Holmes_eric.jpg" /><br />
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<div class="row" id="huang"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5b/Huang_danielle.jpg" /><br />
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<h3>Danielle Huang</h3><br />
Her second consecutive year in iGEM, Danielle has proven time and again that she's invaluable to the world of Biological Engineering. When she's not sitting in one of the many classes she's taking, you'll find her conducting complicated experiments in the lab. Already outclassing the Cornell biology labs by her junior year, Danielle spent 8 months doing an Engineering Co-Op at Johnson & Johnson as well as Regeneron Pharmaceuticals, assisting in process development and antibody therapeutics analysis. Danielle is the person you want to bring with you on a nighttime walk; she will enlighten you about the stars and constellations, in addition to being a great person to hang out with. She is one of our team's highest commodities, being in demand by everyone all the time.<br />
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<div class="row" id="leach"><br />
<div class="nine columns"><br />
<h3>Daniel Leach</h3><br />
His stare is the most efficient selection marker. To transform, bacteria just ask him for plasmids. His pipetting thumb alone has made more progress than lesser scientists’ entire bodies. He doesn’t worry about being sterile; other organisms avoid his work out of respect. The parts registry accepted his genome as a standard part. He once failed a procedure, just to see how it feels. His scent grows <i>E. coli</i> faster than LB. He is the most interesting iGEM competitor in the world. <br />
<br><br><br />
“I don’t always create new plasmids, but when I do, I prefer Biobricks."<br />
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<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/b/b8/Leach_dan.jpg" /><br />
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<div class="row" id="lizarralde"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/0/0c/Lizarralde_rafael.jpg" /><br />
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<div class="nine columns"><br />
<h3>Rafael Lizarralde</h3><br />
Rafael is a member of the latest generation of ninjas, and spends the majority of his time battling creatures from the seventh dimension. Not only has he mastered the art of dandelion decapitation, he also recently learned how to call forth the power of the heavens after many long hours studying rain dance simulations. <br><br><br />
His interactions with other dimensions have left him with an uncanny knack for engineering. Some of his latest creations include the mobius bagel and a controller eerily reminiscent of the iPhone 5, but so dangerous that he won't let lesser mortals even approach the device. Any ideas about what exactly this controller does are mere speculation; however, since its invention Rafael seems to have developed an unhealthy obsession with spirals, and can occasionally be found talking to snails. <br><br><br />
When not trying to save the world from impending doom, Rafael enjoys cuddling with his favorite glow-in-the-dark purple cuttlefish, crushing defenseless plushie Totoros, and tormenting young iGEMers with a gaze that makes even E. coli scream (research pending on how this affects transformation efficiency).<br />
</div><br />
</div><br />
<div class="row" id="luengvarinkul"><br />
<div class="nine columns"><br />
<h3>Oat Luengvarinkul</h3><br />
Portrayed by actor Jesse Eisenberg in the upcoming 2014 film TSN, Episode II, Oat is a Thai architect/computer programmer and internet entrepeneur. The film's production studio offers a spoiler-removed description of his biography/ the movie's plot:<br />
<br />
""Together with his college roommates, Oat launched social networking site ******** from Cornell's dormitory rooms to great success before introducing ******** onto other campuses nationwide and relocating ******** to Palo Alto, California. But his hearty ******** payday would come at a price. Mired in legal disputes, Oat soon discovered that many of those he had befriended on and off ******** during his rise to the top were eager to see him fall. As ex-friends engage Oat in the courtroom for ownership of ********, it appears that the young entrepreneur might have let ********-based greed eclipse his better judgment. Director David Fincher (Seven, Fight Club) offers Oat's early life as an exploration into the meaning of success in the early 21st century from the perspectives of the innovative ******** founders who revolutionized the way we communicate.""<br />
<br />
Also, he dabbles in architecture. And Facebook.<br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Luengvarinkul_oat.jpg" /><br />
</div><br />
</div><br />
<div class="row" id="ly"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7a/Ly_jeffrey.jpg" /><br />
</div><br />
<div class="nine columns"><br />
<h3>Jeffrey Ly</h3><br />
From the moment Jeffrey was born, he knew he was destined to join the Cornell iGEM team. Two years later, he was successfully recruited. His youth isn't all drawback: his energy rivals that of his best friend and mentor, the energizer bunny, and it allows him to be more on top of things than most people. It has also earned him a place on the team as director of all things requiring enthusiasm. His other responsibilities include optimizing his teammates' vacation schedules and cheering up cells recovering from transformation.<br />
You may say he's a dreamer, but he is the only one. </br><br />
Isn't he cool?<br />
</div><br />
</div><br />
<div class="row" id="sarkar"><br />
<div class="nine columns"><br />
<h3>Ritvik Sarkar</h3><br />
Born in Calcutta, India, Ritvik Sarkar is a world renowned Poké-master, best known for conquering the Elite Four with a level-7 Charmander. Growing bored with his constant victories, he chose to pursue a degree in mechanical engineering at Cornell University. As a member of Cornell iGEM, Ritvik can hold his own in the machine shop, and has also been a valuable asset during outreach events. As a Taekwondo black belt, Ritvik is also indispensable at the perpetually rambunctious iGEM social events. Ritvik enjoys listening to music and playing the cello, for which he also composes pieces. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/9/9d/Sarkar_ritvik.jpg" /><br />
</div><br />
</div><br />
<div class="row" id="sennet"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/a/af/Sennett_mac.jpg" /><br />
</div><br />
<div class="nine columns"><br />
<h3>Mac Sennett</h3><br />
Mac came to CUGEM by an unidentified aircraft that was claimed by Ithaca’s gorges. In order to repair his ship and travel back to his own galaxy, Mac joined our team as the Dry Lab leader in order to have access to the machine shop, where he quickly displayed his other worldly skills. Although initially intent on repairing his aircraft and returning home, Mac changed his mind upon realizing how amazing synthetic biology was.<br />
</br></br><br />
When not leading the fabrication aspect of the project, Mac can be seen holding his own with the best of biologists. Despite his suboptimal pouring technique which he uses to discourage suspicion, many of us are convinced that Mac may be coercing his bacteria to follow his will with his supernatural powers.<br />
</br></br><br />
Once an intergalactic mercenary, Mac has continued his desire for excitement by moonlighting as Ithaca’s own superhero. When not saving freshman from ill-advised gorge jumping and the bipolar weather, Mac can be seen answering the prayers of his fellow iGEM members, once appearing out of nowhere to give his stranded members a ride, delivering his catch phrase, “come to daddy.”<br />
</br></br><br />
For the moment, Mac has decided to settle down in Ithaca to pursue his interests in neurobiology and biochemistry. For the sake of mankind, let’s hope he decides to stay here forever. <br />
</div><br />
</div><br />
<div class="row" id="sharma"><br />
<div class="nine columns"><br />
<h3>Prashant Sharma</h3><br />
Here we have spotted the rare Prashant Sharma in his natural habitat. Known as Shawn by the natives of these bleached-white halls, this elusive species is most easily sighted when mingling with others of its kind. Unlike previously examined organisms, those of the Prashant genus are capable of an untroubled manner granting them a unique niche in this high-stress ecosystem. However, also characteristic of the Shawn is an innate sense of humor that both its prospective predators and prey find startling. While researchers continue to study this enigmatic creature, there is much debate over the ethics and legality of their use as laboratory assistants,<br />
despite their seeming acuity at it. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Sharma_prashant.jpg" /><br />
</div><br />
</div><br />
<div class="row" id="simpson"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/1/12/Simpson_mark.jpg" /><br />
</div><br />
<div class="nine columns"><br />
<h3>Mark Simpson</h3><br />
Knighted as “the God of Electrocompetent Stocks,” Mark has aroused a growing religion among iGEMers worshipping his ability to create infinite stocks with the bat of an eye. He has astounded his followers with his ability to eat red bell peppers raw and his Pillsbury doughboy impression will leave you giggling on the outside, but crying on the inside…out of happiness of course. After winging…I mean, graduating in 3 years, Mark acquired the nickname “Turbo!”, also reflecting his godliness in the lab. Mark is a valuable asset to our team, having trademarked several widely employed lab techniques such as hand centrifugation and mouth pressurized column chromatography. His sense of humor brightens the room, providing zesty comedic relief to our meetings. "Meeting adjourned!" <br />
</div><br />
</div><br />
<div class="row" id="soong"><br />
<div class="nine columns"><br />
<h3>Christine Soong</h3><br />
Christine is from Ohio. Enough said. But wait, there’s more… </br><br />
In the U.S. government’s time of need, they turned to Christine. She did not let them down. Reluctant to leave the iGEM team, but knowing that country comes first, Christine heroically chose to spend her summer at a top-secret military intelligence base. She masquerades as a summer intern at NASA, but in actuality she is a member of the CIA. Of course if you ask her she would deny it; that is the mark of a true CIA operative. Christine spends her days intercepting and decoding satellite communications from not-so-friendly nations, and has prevented two world wars to date. An electronics genius and computer whiz, Christine is extremely talented, but that is only evident through her work, she’s quite humble. An animal lover, she plans to own 101 dalmations after she graduates Cornell in under four years. When she's not working, Christine enjoys running, kayaking, and other outdoor activities. Just as she stepped in to save the U.S., she will return in the fall to make sure the CUGEM team is a success. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/6/68/Soong_christine.jpg" /><br />
</div><br />
</div><br />
<div class="row" id="spassibojko"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/Spassibojko_olya.jpg" /><br />
</div><br />
<div class="nine columns"><br />
<h3>Olya Spassibojko</h3><br />
While many are left spinning like a centrifuge at 15,000rpm and scratching their heads as they attempt to follow our mycelium-like meetings where team members never fail to branch off each other’s ideas, Olya calmly captures the pertinent points in her impeccably kept minutes. When not typing and uploading the team’s never ending stream of minutes, she can be found in the wetlab running PCRs and secretly wishing that the loading dye was a few shades lighter and turqoise in color just like everything else she owns. Have we mentioned that the “Bio-philic Olya” absolutely adores her fluffy, hypoallergenic, turquoise kitty which listens to Anberlin too? <br />
</div><br />
</div><br />
<div class="row" id="su"><br />
<div class="nine columns"><br />
<h3>Tina Su</h3><br />
TinamiSu </br><br />
</br><br />
2 Whole Dill Pickles </br><br />
A cup of oatmeal </br><br />
1 cup Plain Greek Yogurt </br><br />
3 thirds of a cup roasted chili pepper hummus </br><br />
.786 baby carrots </br><br />
A dollop of mayonnaise </br><br />
1 aliquot of honey </br><br />
</br><br />
Sweet, Delicious, and Expensive. TinamiSu is a must try. Although very sweet, it certainly doesn’t lack a bit of a kick. Acclaimed for its creamy texture, it is sure to get all your friends talking—maybe even a little too much. Regardless, TinamiSu is always tasteful and a fun dish. Served both hot or cold, you know you can turn to it on your happy days, your difficult days, or even your rainy days. </br><br />
This dish is best consumed very slowly with a pipettor.<br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5b/Su_tina.jpg" /><br />
</div><br />
</div><br />
<div class="row" id="sureka"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/b/b7/Sureka_swati.jpg" /><br />
</div><br />
<div class="nine columns"><br />
<h3>Swati Sureka</h3><br />
When we accepted her onto the team last year, Swati was an unassuming freshman. Little did we know she was a ticking time-bomb just waiting to go off. Her rapid rise to power makes perfect sense in retrospect: she is well-versed in four different martial arts and has received training from two separate intelligence agencies. She has no need for competent cells because she has developed her own martial art, clonejutsu, which allows her to punch, kick, or elbow DNA directly into the cells of any organism. She possesses great strength, cunning, and ruthlessness, and will be the most difficult adversary you ever face.<br />
</div><br />
</div><br />
<div class="row" id="toyoda"><br />
<div class="nine columns"><br />
<h3>Yoshiko Toyoda</h3><br />
Yoshi is the muse of our iGEM team. She comes in the early mornings to set up PCRs to prepare everyone else’s day in lab. Her awe-inspiring penmanship on primers and mini-preps brings clarity to our otherwise chaotic assembly of reagents. Faster than a green dinosaur or a trusty mid-sized automobile, Yoshi works with great diligence and grace, even when under pressure. When she is done for the day, she relaxes with some of the finer works of culture, such as her history readings or Japanese soap operas. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/5/54/Toyoda_yoshi.jpg" /><br />
</div><br />
</div><br />
<div class="row" id="wang"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Wang_lydia.jpg" /><br />
</div><br />
<div class="nine columns"><br />
<h3>Lydia Wang</h3><br />
Still using Snap Chat? That is going to be outdated soon for college students. Our electrical & computer engineering genius—Lydia—is working on developing super advanced mobile apps for the iPhone 11 that can literally read your mind and plan your life for the next 50 years (…or more). As a co-op in GE Intelligent Platforms in 2013, Lydia has already started her master plan. She joined iGEM in the spring of 2012 to gather information on what smart people in Cornell do in their daily lives for her project, but was disappointed to find that they are actually not robots, but a bunch of kids playing with bacteria and pipettes. So she decided to stay and help out with the Dry Lab, while secretly incorporating microcontrollers in whatever systems the team develops to monitor and control iGEMers, slowly turning them into real robots by the end of 2013. Even though a few people on the team know of her plan, they are more than happy to accept whatever Lydia put into the Dry Lab components because they make the systems work like magic. And ultimately, they accept the fact that they will become robots someday.<br />
</div><br />
</div><br />
<div class="row last-ele" id="wheeler"><br />
<div class="nine columns"><br />
<h3>Kyle Wheeler</h3><br />
Kyle Wheeler is Cornell iGEM's resident cyborg. After failing to ride a century as a wee babe, Kyle, in his infinite infant wisdom, had an epiphany: to succeed, he must genetically engineer himself to become one with the machine. Thus began his interest in iGEM. Surrounded in a shroud of mystery, Kyle tries to protect his secret identity through seemingly innocuous activities like coding and eating lots of organic produce. Hailing from a long line of iGEM Risley-ites, he keeps himself in physical shape by casually biking 40+ miles around lakes and mentally fit by perfoming wet lab vodoo and concocting business plans on the sponsorship subteam. Ladies, try to keep up with this man's genius, but don't worry too much - no matter how slow you go or whatever torrential thunderstorm you find yourself in, Kyle's the kind of guy that rides by your side. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4c/Wheeler_kyle.jpg" /><br />
</div><br />
</div><br />
<br><br />
<div id="faculty" class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Faculty Advisors</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ea/Cornell_turgeon.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Dr. B. Gillian Turgeon - Plant Pathology and Plant-Microbe Biology</h3><br />
Gillian Turgeon is a fungal geneticist studying molecular mechanisms by which fungi cause disease on plants and the evolution of diverse reproductive strategies. Both are recognition questions. For pathogenicity the issue is how cells from different organisms, i.e., the fungus and the plant host, communicate with each other to cause or prevent disease. For sexual reproductive development, fungi either require a partner of opposite mating type to mate or can self-mate, thus the issue is how fungal cells recognize self from non-self. Using the filamentous ascomycete plant pathogen Cochliobolus heterostrophus and close relatives for comparison, the Turgeon lab pioneered molecular genetic, genomic, and phylogenomic tools for filamentous fungi and has deployed these to illuminate the roles of secondary metabolites in plant pathogenesis and host specificity, reproductive development, iron metabolism and oxidative stress, and niche adaptation.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Dr. Xiling Shen - Electrical and Computer Engineering</h3><br />
Dr. Xiling Shen has been an assistant professor in the School of Electrical and Computer Engineering at Cornell University since August 2009.<br />
<br><br><br />
Born in Shanghai, China, Dr. Xiling Shen went on to receive his BS and MS degree from the Electrical Engineering Department of Stanford University in 2001. He then worked at Barcelona Design Inc. for two years, specializing in analog circuit design and optimization, before joining Professor Mark Horowtiz' research group in the Electrical Engineering Department at Stanford in 2003. In the first two years of his PhD, he collaborated with Professor Joseph Kahn on using adaptive spatial equalization to compensate modal dispersion in multimode fibers. From 2005 to 2008, he worked with Professor Harley McAdams, Professor Lucy Shapiro, and Professor David Dill on modeling and analyzing the asymmetric division of Caulobacter crescentus. Xiling’s postdoctoral work focused on synthetic biology with Dr. Adam Arkin in Bioengineering at UC Berkeley prior to joining the faculty at Cornell University’s School of Electrical and Computer Engineering.<br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/2/29/Cornell_SHEN.jpg"><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/2/26/Cornell_ARCHER.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Dr. Shivaun Archer - Biomedical Engineering</h3><br />
Dr. Shivaun Archer is a Senior Lecturer in charge of the Biomedical Engineering Undergraduate Instructional Laboratories. She designs and teaches undergraduate instructional labs for five biomedical engineering courses: BME 131, BME 301, BME 302, BME 401, and BME 402. The labs are designed to illustrate the course material and bring research to undergraduate education whilst exposing students to cutting edge technology and research methodology. A significant emphasis in all the labs is biomedical nanotechnology. Each of the five courses has a hands-on lab module that focuses specifically on nanobiotechnology. Overall, the lab modules enhance the hands-on training of Cornell students in the areas of microfabrication, microfluidics, biosensors, nano/microbiotechnology, and drug delivery. In recognition of her efforts in undergraduate education, Dr. Archer has received a prestigious College of Engineering Teaching award.<br />
<br><br><br />
Before coming to Cornell, Dr. Archer worked for five years at Lynntech, Inc. a small research company specializing in biotechnology, biomaterials, chemical and biological sensors, medical biotechnology, and environmental remediation. Her work on wastewater treatment for long term space missions resulted in her receiving two NASA Inventions Space Act Awards. She also holds a joint appointment as a Research Associate in the School of Chemical and Biomolecular Engineering. Her research interests include nanobiotechnology and tissue engineering.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Dr. Matthew DeLisa - Chemical Engineering</h3><br />
Matthew DeLisa received his B.S. in Chemical Engineering from the University of Connecticut in 1996; his Ph.D. in Chemical Engineering from the University of Maryland in 2001; and did postdoctoral work at the University of Texas-Austin, Department of Chemical Engineering. DeLisa joined the Department of Chemical and Biomolecular Engineering at Cornell University as an assistant professor in 2003 and was promoted to associate professor in 2009. He recently served as a Gastprofessur at ETH Zürich in the Institut für Mikrobiologie.<br />
<br><br><br />
Professor DeLisa's research focuses on understanding and controlling the molecular mechanisms underlying protein biogenesis -- folding and assembly, membrane translocation and post-translational modifications -- in the complex environment of a living cell. His contributions to science and engineering include the invention of numerous commercially important technologies for facilitating the discovery, design and manufacturing of human drugs and seminal discoveries in the areas of cellular protein folding and protein translocation. DeLisa has received several awards for his work including an NSF CAREER award, a NYSTAR Watson Young Investigator award, a Beckman Foundation Young Investigator award, an Office of Naval Research Young Investigator award, and a NYSTAR Distinguished Faculty Award. He was also named one of the top 35 young innovators (TR35) by MIT's Technology Review in 2005 and was selected as the inaugural recipient of the Wiley-Blackwell Biotechnology and Bioengineering Daniel I.C. Wang award, which honors a distinguished young researcher in this field. Most recently, he was honored with a Cornell Provost's Award for Distinguished Scholarship and was the recipient of the Young Investigator Award from the American Chemical Society's BIOT division.<br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1e/Cornell_DELISA.jpg"><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e1/Cornell_lucks.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Dr. Julius B. Lucks - Chemical and Biomolecular Engineering</h3><br />
Julius B. Lucks is Assistant Professor of Chemical and Biomolecular Engineering at Cornell University, and a James C. and Rebecca Q. Morgan Sesquicentennial Faculty Fellow. After attending the North Carolina School of Science and Mathematics for high school, he became an undergraduate at the University of North Carolina at Chapel Hill where he performed research in organic synthesis and the application of density functional theory to studying the electronic properties of atoms and molecules as a Goldwater Scholar. After graduating with a BS in Chemistry, he spent a summer working with Robert Parr before obtaining an M. Phil. in Theoretical Chemistry at Cambridge University as a Churchill Scholar. As a Hertz Fellow at Harvard University, he researched problems in theoretical biophysics including RNA folding and translocation, viral capsid structure and viral genome organization, under David R. Nelson. As a Miller Fellow at UC Berkeley in the laboratory of Adam P. Arkin, he engineered versatile RNA-sensing transcriptional regulators that can be easily reconfigured to independently regulate multiple genes, logically control gene expression, and propagate signals as RNA molecules in gene networks. He also lead the team that developed SHAPE-Seq, an experimental technique that utilizes next generation sequencing for probing RNA secondary and tertiary structures of hundreds of RNAs in a single experiment.<br />
<br><br><br />
Professor Lucks’ research combines both experiment and theory to ask fundamental questions about the design principles that govern how RNAs fold and function in living organisms, and how these principles can be used to engineer biomolecular systems, and open doors to new medical therapeutics.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Dr. Bruce Land - Electrical and Computer Engineering</h3><br />
Bruce Land is a Senior Lecturer in Electrical and Computer Engineering at Cornell. He teaches three courses in ECE and advises masters of engineering projects in ECE and Biomedical Engineering. When time allows, he does some neural modeling and spike train analysis. He has been in this position since 1998.<br />
<br><br><br />
Land received a BS in physics from Harvey Mudd College in 1968 and a Ph.D. in neurobiology from Cornell University in 1976 . He was a Muscular Dystrophy Association postdoc in NBB at Cornell for three years, then a lecturer in NBB for seven years. During this time he worked with Miriam Salpeter on the coupling of activity at the vertebrate neuromuscular junction, both experimentally and by computer modeling. In 1987 he moved to the Cornell Theory Center as a computational research associate, then started supporting graphics and animation. He was visualization project leader at the CTC from 1989 to 1998. From 1992 to 1998 he taught an introductory computer graphics course in Computer Science at Cornell. From 1998 to 2007 he taught computer programming and electronics courses in NBB and was a Senior Research Associate in Neurobiology and Behavior.<br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/BRUCE.jpg"><br />
</div><br />
</div><br />
<br><br />
<div id="gradadvisors" class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Graduate and Post-Grad Advisors</h2><br />
<br />
</div><br />
</div><br />
<div class="row last-ele"><br />
<div class="twelve columns"><br />
<ul class="grd-list block-grid three-up" style="text-align:center;"><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8d/Cornell_dongliang.jpg"><br />
</div><br />
<h5 class="centered">Dongliang Wu</h5><br />
Turgeon Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e2/Cornell_bradford.jpg"><br />
</div><br />
<h5 class="centered">Bradford Condon</h5><br />
Turgeon Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/12/Taylor_small.jpg"><br />
</div><br />
<h5 class="centered">Taylor Stevenson</h5><br />
DeLisa Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/Jennifer_Fricke.jpg"><br />
</div><br />
<h5 class="centered">Jennifer Fricke</h5><br />
Clark Lab <br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/7/79/Daniel_Kolbin.jpg"><br />
</div><br />
<h5 class="centered">Daniel Kolbin</h5><br />
Clark Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1a/Cornell_aravind.png"><br />
</div><br />
<h5 class="centered">Aravind Natarajan</h5><br />
DeLisa Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/7/70/Cornell_jason.jpg"><br />
</div><br />
<h5 class="centered">Jason Kahn</h5><br />
Luo Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/5/50/Cornell_devin.jpg"><br />
</div><br />
<h5 class="centered">Devin Doud</h5><br />
Angenent Lab<br />
</li><br />
<li><br />
<div style="width:166px;margin:auto;"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cd/Cornell_matthew.JPG"><br />
</div><br />
<h5 class="centered">Matthew Russell</h5><br />
March Lab<br />
</li><br />
</ul><br />
</div><br />
</div><br />
<br><br />
<div id="corporate" class="row"><br />
<div class="twelve columns"><br />
<h2 class="centered">Corporate Advisors</h2><br />
</div><br />
</div><br />
<div class="row"><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/9/97/CornellGavin.jpg"><br />
</div><br />
<div class="nine columns"><br />
<h3>Gavin McIntyre - Ecovative Design</h3><br />
Gavin McIntyre is the co-founder and Chief Scientist of Ecovative Design. He received a dual B.S. in Mechanical Engineering and Product Design from Rensselaer Polytechnic Institute. Since the company's start in 2007, he has led all material and biological process development. <br />
<br><br><br />
Gavin has served as the principle investigator on grants received from the US EPA, the USDA, the NSF, Empire State Development, and the New York State Energy Research and Development Authority. He serves on the board of directors for Ecovative, the NYS Pollution Prevention Institute, and the Rensselaer Center for Automation Technology and Systems. He has also served on Technology Roundtables hosted by former NYS Governor David Paterson, NYS Governor Cuomo, the US DoE, the NSF, and the US EPA.<br />
</div><br />
</div><br />
<div class="row"><br />
<div class="nine columns"><br />
<h3>Sam Harrington - Ecovative Design</h3><br />
Sam Harrington is Ecovative's Marketing Manager. He graduated from Rensselaer Polytechnic Institute with a degree in Mechanical Engineering and Product Design & Innovation. After joining the firm shortly after it was established, Sam spent two years as Ecovative's Environmental Director. Sam has conducted Life Cycle Assessments of the production system to inform engineering decisions and minimize Ecovative's environmental footprint. Sam now leads the marketing and sales team at Ecovative and has worked with several Fortune 500 customers to develop award winning sustainable packaging solutions. He is a two time winner of the Change the World Challenge and a winner in the international Dyson Eye for Why Design Competition. <br />
</div><br />
<div class="three columns"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5b/CornellHarrington.jpg"><br />
</div><br />
</div><br />
<br />
</div><br />
</div><br />
</body><br />
</html></div>R.Lizarraldehttp://2013.igem.org/File:CornellHarrington.jpgFile:CornellHarrington.jpg2013-09-28T01:26:56Z<p>R.Lizarralde: </p>
<hr />
<div></div>R.Lizarraldehttp://2013.igem.org/File:CornellGavin.jpgFile:CornellGavin.jpg2013-09-28T01:25:48Z<p>R.Lizarralde: </p>
<hr />
<div></div>R.Lizarraldehttp://2013.igem.org/Team:Cornell/javascripts/notebookTeam:Cornell/javascripts/notebook2013-09-28T01:17:32Z<p>R.Lizarralde: </p>
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["Tim", "Tim Abbot", <br />
"https://static.igem.org/mediawiki/2013/6/62/Abbott_tim.jpg", <br />
"Wetlab member", <br />
"https://2013.igem.org/Team:Cornell/team/bios#abbot"<br />
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["Hannah", "Hannah Ajmani", <br />
"https://static.igem.org/mediawiki/2013/1/18/Ajmani_hannah.jpg", <br />
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["Eric Appel", "Eric Appel", <br />
"https://static.igem.org/mediawiki/2013/2/28/Appel_e.jpg", <br />
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["Ryan", "Ryan Ashley", <br />
"https://static.igem.org/mediawiki/2013/e/ea/Ashley_ryan.jpg", <br />
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],<br />
["Nupur", "Nupur Bhatt", <br />
"https://static.igem.org/mediawiki/2013/c/cf/Bhatt_nupur.jpg", <br />
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],<br />
["Arun", "Arun Chakravorty", <br />
"https://static.igem.org/mediawiki/2013/c/c6/Chakravorty_arun.jpg", <br />
"Wetlab taskforce leader, presenter, and overall awesome guy", <br />
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],<br />
["Jonlin", "Jonlin Chen", <br />
"https://static.igem.org/mediawiki/2013/4/49/Chen_jonlin.jpg", <br />
"Wetlab member and miniprep master", <br />
"https://2013.igem.org/Team:Cornell/team/bios#chen"<br />
],<br />
["Rebecca", "Rebecca Chew", <br />
"https://static.igem.org/mediawiki/2013/c/c0/Chew_rebecca.jpg", <br />
"Drylab member and wetlab member", <br />
"https://2013.igem.org/Team:Cornell/team/bios#chew"<br />
],<br />
["Sharlene", "Sharlene Dong", <br />
"https://static.igem.org/mediawiki/2013/1/17/Dong_sharlene.jpg", <br />
"Wetlab taskforce leader", <br />
"https://2013.igem.org/Team:Cornell/team/bios#dong"<br />
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["Sara", "Sara Gregg", <br />
"https://static.igem.org/mediawiki/2013/e/e4/Gregg_sara.jpg", <br />
"Drylab member and wetlab member", <br />
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],<br />
["Alex", "Alex Han", <br />
"https://static.igem.org/mediawiki/2013/4/4e/Han_alex.jpg", <br />
"Wetlab taskforce leader", <br />
"https://2013.igem.org/Team:Cornell/team/bios#han"<br />
],<br />
["Eric", "Eric Holmes", <br />
"https://static.igem.org/mediawiki/2013/9/94/Holmes_eric.jpg", <br />
"Wetlab taskforce leader and presenter", <br />
"https://2013.igem.org/Team:Cornell/team/bios#holmes"<br />
],<br />
["Danielle", "Danielle Huang", <br />
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"Human practices and outreach coordinator", <br />
"https://2013.igem.org/Team:Cornell/team/bios#huang"<br />
],<br />
["Dan", "Daniel Leach", <br />
"", <br />
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["Rafael", "Rafael Lizarralde", <br />
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["Oat", "Oat Luengvarinkul", <br />
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["Jeffrey", "Jeffrey Ly", <br />
"https://static.igem.org/mediawiki/2013/7/7a/Ly_jeffrey.jpg", <br />
"Wetlab member and energizer bunny", <br />
"https://2013.igem.org/Team:Cornell/team/bios#ly"<br />
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["Ritvik", "Ritvik Sarkar", <br />
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"Drylab member.", <br />
"https://2013.igem.org/Team:Cornell/team/bios#sarkar"<br />
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["Mac", "Mac Sennet", <br />
"https://static.igem.org/mediawiki/2013/a/af/Sennett_mac.jpg", <br />
"Drylab leader.", <br />
"https://2013.igem.org/Team:Cornell/team/bios#sennet"<br />
],<br />
["Prashant", "Prashant Sharma", <br />
"https://static.igem.org/mediawiki/2013/d/d5/Sharma_prashant.jpg", <br />
"Wetlab member and energizer bunny", <br />
"https://2013.igem.org/Team:Cornell/team/bios#sharma"<br />
],<br />
["Mark", "Mark Simpson", <br />
"https://static.igem.org/mediawiki/2013/1/12/Simpson_mark.jpg", <br />
"Wetlab member and energizer bunny", <br />
"https://2013.igem.org/Team:Cornell/team/bios#simpson"<br />
],<br />
["Christine", "Christine Soong", <br />
"https://static.igem.org/mediawiki/2013/6/68/Soong_christine.jpg", <br />
"Drylab member.", <br />
"https://2013.igem.org/Team:Cornell/team/bios#soong"<br />
],<br />
["Olya", "Olya Spassibojko", <br />
"https://static.igem.org/mediawiki/2013/c/ca/Spassibojko_olya.jpg", <br />
"Wetlab taskforce leader", <br />
"https://2013.igem.org/Team:Cornell/team/bios#spassibojko"<br />
],<br />
["Tina", "Tina Su", <br />
"https://static.igem.org/mediawiki/2013/5/5b/Su_tina.jpg", <br />
"Wetlab member and sponsorship team leader", <br />
"https://2013.igem.org/Team:Cornell/team/bios#su"<br />
],<br />
["Swati", "Swati Sureka", <br />
"https://static.igem.org/mediawiki/2013/b/b7/Sureka_swati.jpg", <br />
"The Boss", <br />
"https://2013.igem.org/Team:Cornell/team/bios#sureka"<br />
],<br />
["Yoshi", "Yoshiko Toyoda", <br />
"https://static.igem.org/mediawiki/2013/5/54/Toyoda_yoshi.jpg", <br />
"Wetlab member", <br />
"https://2013.igem.org/Team:Cornell/team/bios#toyoda"<br />
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["Yoshiko", "Yoshiko Toyoda", <br />
"https://static.igem.org/mediawiki/2013/5/54/Toyoda_yoshi.jpg", <br />
"Wetlab member", <br />
"https://2013.igem.org/Team:Cornell/team/bios#toyoda"<br />
],<br />
["Lydia", "Lydia Wang", <br />
"https://static.igem.org/mediawiki/2013/3/35/Wang_lydia.jpg", <br />
"Drylab member.", <br />
"https://2013.igem.org/Team:Cornell/team/bios#wang"<br />
],<br />
["Kyle", "Kyle Wheeler", <br />
"https://static.igem.org/mediawiki/2013/4/4c/Wheeler_kyle.jpg", <br />
"Wetlab member.", <br />
"https://2013.igem.org/Team:Cornell/team/bios#wheeler"<br />
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<!-- PLASMIDS --><br />
["pAK13AA", "pAK13AA", "", "PT7 + crtE in pSB1AK8", ""],<br />
["pAK13AB", "pAK13AB", "", "PT7 + crtB in pSB1AK8", ""],<br />
["pAK13AC", "pAK13AC", "", "PT7 + crtI in pSB1AK8", ""],<br />
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["pC13AI", "pC13AI", "", "lox site in pSB1C3", ""],<br />
["pA13AJ", "pA13AJ", "", "lox site in pSB1A2", ""],<br />
["pC13AK", "pC13AK", "", "mRFP (coding sequence only) in pSB1C3", ""],<br />
["pK13AL", "pK13AL", "", "mRFP (coding sequence only) in pSB2K3", ""],<br />
["pA13AM", "pA13AM", "", "Strongest Anderson constitutive promoter + mRFP in BBa_J61002", ""],<br />
["pAK13AN", "pAK13AN", "", "PT7 + GFP in pSB1AK8", ""],<br />
["pAK13AO", "pAK13AO", "", "PT7 + mRFP in pSB1AK8", ""],<br />
["pAK13AP", "pAK13AP", "", "PT7 + bar in pSB1AK8", ""],<br />
["pC13AQ", "pC13AQ", "", "PT7 + GFP in pSB1C3", ""],<br />
["pC13AR", "pC13AR", "", "PT7 + mRFP in pSB1C3", ""],<br />
["pC13AS", "pC13AS", "", "PT7 + bar in pSB1C3", ""],<br />
["pC13AT", "pC13AT", "", "PtrpC + GFP in pSB1C3", ""],<br />
["pC13AU", "pC13AU", "", "PtrpC + mRFP in pSB1C3", ""],<br />
["pC13AV", "pC13AV", "", "PtrpC + bar in pSB1C3", ""],<br />
["pA13AW", "pA13AW", "", "Anderson promoter + T7 polymerase from BBa_J61002", ""],<br />
["pK13AX", "pK13AX", "", "Anderson promoter + T7 polymerase in pSB2K3", ""],<br />
["pC13AY", "pC13AY", "", "PtrpC + T7 polymerase in pSB1C3", ""],<br />
["pC13AZ", "pC13AZ", "", "Anderson promoter + T7 polymerase in pSB1C3", ""],<br />
<br />
["pC13BA", "pC13BA", "", "afp1 in pSB1C3", ""],<br />
["pAK13BB", "pAK13BB", "", "PT7 + nptII biobrick in pSB1AK8", ""],<br />
["pC13BC", "pC13BC", "", "PT7 + nptII biobrick in pSB1C3", ""],<br />
["pC13BD", "pC13BD", "", "PtrpC + crtI in pSB1C3", ""],<br />
["pA13BE", "pA13BE", "", "PpelA, flanked by KpnI and BamHI cutsites in pHPG", ""],<br />
["pC13BF", "pC13BF", "", "PpelA in pSB1C3", ""],<br />
["pC13BG", "pC13BG", "", "A. nidulans PgpdA in pSB1C3", ""],<br />
["pC13BH", "pC13BH", "", "G. lucidum PgpdA in pSB1C3", ""],<br />
["pC13BI", "pC13BI", "", "G. lucidum PgpdA biobrick in pSB1C3", ""],<br />
["pC13BJ", "pC13BJ", "", "100bp left region of homology of G. lucidum PgpdA in pSB1C3", ""],<br />
["pC13BK", "pC13BK", "", "200bp left region of homology of G. lucidum PgpdA in pSB1C3", ""],<br />
["pC13BL", "pC13BL", "", "100bp right region of homology of G. lucidum PgpdA in pSB1C3", ""],<br />
["pC13BM", "pC13BM", "", "200bp right region of homology of G. lucidum PgpdA in pSB1C3", ""],<br />
["pC13BN", "pC13BN", "", "lox [cutsite 1] [cutsite 2] lox in pSB1C3", ""],<br />
["pC13BO", "pC13BO", "", "short homology regions flanking two unique cutsites in pSB1C3", ""],<br />
["pC13BP", "pC13BP", "", "long homology regions flanking two unique cutsites in pSB1C3", ""],<br />
["pC13BQ", "pC13BQ", "", "A. nidulans PgpdA + nptII biobrick in pSB1C3", ""],<br />
["pC13BR", "pC13BR", "", "A. nidulans PgpdA + hph biobrick in pSB1C3", ""],<br />
["pC13BS", "pC13BS", "", "G. lucidum PgpdA biobrick + nptII biobrick in pSB1C3", ""],<br />
["pC13BT", "pC13BT", "", "G. lucidum Pgpd biobrick + hph biobrick in pSB1C3", ""],<br />
["pAK13BU", "pAK13BU", "", "PT7 + hph biobrick in pSB1AK8", ""],<br />
["pC13BV", "pC13BV", "", "PT7 + hph biobrick in pSB1C3", ""],<br />
["pA13BW", "pA13BW", "", "CMV Cre in pBS185", ""],<br />
["pC13BX", "pC13BX", "", "Cre recombinase in pSB1C3", ""],<br />
["pC13BY", "pC13BY", "", "PpelA + Cre in pSB1C3", ""],<br />
["pC13BZ", "pC13BZ", "", "PpelA + GFP in pSB1C3", ""],<br />
<br />
["pC13CA", "pC13CA", "", "PpelA + mRFP in pSB1C3", ""],<br />
["pC13CB", "pC13CB", "", "PT7 + GFP + PtrpC + nptII biobrick in pSB1C3", ""],<br />
["pC13CC", "pC13CC", "", "PtrpC + nptII biobrick + TtrpC in pSB1C3", ""],<br />
["pC13CD", "pC13CD", "", "PtrpC + hph biobrick in pSB1C3", ""],<br />
["pC13CE", "pC13CE", "", "PtrpC + hph biobrick + TtrpC in pSB1C3", ""],<br />
["pC13CF", "pC13CF", "", "A. nidulans PgpdA + GFP in pSB1C3", ""],<br />
["pC13CG", "pC13CG", "", "A. nidulans PgpdA + mRFP in pSB1C3", ""],<br />
["pC13CH", "pC13CH", "", "G. lucium PgpdA + GFP in pSB1C3", ""],<br />
["pC13CI", "pC13CI", "", "G. lucidum PgpdA + mRFP in pSB1C3", ""],<br />
["pC13CJ", "pC13CJ", "", "Limonene synthase in pSB1C3", ""],<br />
["pAK13CK", "pAK13CK", "", "PT7 + Limonene Synthase in pSB1AK8", ""],<br />
["pC13CL", "pC13CL", "", "Cht1_2 in pSB1C3", ""],<br />
["pC13CM", "pC13CM", "", "PpelA + Cht1_2 in pSB1C3", ""],<br />
["pS13CN", "pS13CN", "", "Lambda Holin in BBa_K112950", ""],<br />
["pC13CO", "pC13CO", "", "Lambda Holin in pSB1C3", ""],<br />
["pC13CP", "pC13CP", "", "PpelA + Lambda Holin in pSB1C3", ""],<br />
["pAK13CQ", "pAK13CQ", "", "PT7 + rbs + crtE in pSB1AK8", ""],<br />
["pAK13CR", "pAK13CR", "", "PT7 + rbs + crtB in pSB1AK8", ""],<br />
["pAK13CS", "pAK13CS", "", "PT7 + rbs + crtI in pSB1AK8", ""],<br />
["pAK13CT", "pAK13CT", "", "PT7 + rbs + crtY in pSB1AK8", ""],<br />
["pC13CU", "pC13CU", "", "PT7 + rbs + crtE in pSB1C3", ""],<br />
["pC13CV", "pC13CV", "", "PT7 + rbs + crtB in pSB1C3", ""],<br />
["pC13CW", "pC13CW", "", "PT7 + rbs + crtI in pSB1C3", ""],<br />
["pC13CX", "pC13CX", "", "PT7 + rbs + crtY in pSB1C3", ""],<br />
["pAK13CY", "pAK13CY", "", "PT7 + rbs + GFP in pSB1AK8", ""],<br />
["pA13CZ", "pA13CZ", "", "Strong RBS in pSB1A2", ""],<br />
<br />
["pAK13DA", "pAK13CA", "", "PT7 + rbs + mRFP in pSB1AK8", ""],<br />
["pA13DB", "pA13DB", "", "RBS + GFP in pSB1A2", ""],<br />
["pA13DC", "pA13DC", "", "RBS + mRFP in pSB1A2", ""],<br />
["pC13DD", "pC13DD", "", "PT7 + limonene synthase in pSB1C3", ""],<br />
["pC13DE", "pC13DE", "", "lox + PtrpC + nptII biobrick + lox in pSB1C3", ""],<br />
["pC13DF", "pC13DF", "", "lox + A. nidulans PgpdA + nptII biobrick + lox in pSB1C3", ""],<br />
["pC13DG", "pC13DG", "", "lox + A.nidulans PgpdA + hph biobrick + lox in pSB1C3", ""],<br />
["pC13DH", "pC13DH", "", "lox + PtrpC + hph biobrick + lox in pSB1C3", ""],<br />
["pC13DI", "pC13DI", "", "PtrpC + hph biobrick + GFP in pSB1C3", ""],<br />
["pC13DJ", "pC13DJ", "", "PT7 + crtB + PT7 + crtI in pSB1C3", ""],<br />
["pC13DK", "pC13DK", "", "PT7 + crtE + PT7 + crtB + PT7 + crtI in pSB1C3", ""],<br />
["pC13DL", "pC13DL", "", "PT7 + crtE + PT7 + crtB + PT7 + crtI + PT7 + nptII biobrick in pSB1C3", ""],<br />
["pC13DM", "pC13DM", "", "PT7 + rbs + crtB + PT7 + rbs + crtI in pSB1C3", ""],<br />
["pC13DN", "pC13DN", "", "PT7 + rbs + crtE + PT7 + rbs + crtB + PT7 + rbs + crtI in pSB1C3", ""],<br />
["pC13DO", "pC13DO", "", "G. lucidum PgpdA + crtE in pSB1C3", ""],<br />
["pC13DP", "pC13DP", "", "A. nidulans PgpdA + crtB in pSB1C3", ""],<br />
["pC13DQ", "pC13DQ", "", "A. nidulans PgpdA + crtB + PtrpC + crtI in pSB1C3", ""],<br />
["pC13DR", "pC13DR", "", "G. lucidum PgpdA + crtE + A. nidulans PgpdA + crtB + PtrpC + crtI in pSB1C3", ""],<br />
["pC13A", "pC13A", "", "crtE + crtI + crtB in pSB1C3", ""],<br />
["pC13B", "pC13B", "", "crtY in PSB1C3", ""],<br />
["pA13C", "pA13C", "", "T7 promoter and terminator in pIVEX2.3d", ""],<br />
["pAK13D", "pAK13D", "", "T7 promoter in PSB1AK8", ""],<br />
["pC13E", "pC13E", "", "T7 polymerase in pSB1C3", ""],<br />
["pC13F", "pC13F", "", "mRFP in pSB1C3", ""],<br />
["pAh13G", "pAh13G", "", "PtrpC + hph + TtrpC in pUCATPH", ""],<br />
["pAg13H", "pAg13H", "", "Pgpd (C.h.) + nptII in pNG", ""],<br />
["pC13I", "pC13I", "", "crtE in pSB1C3", ""],<br />
["pC13J", "pC13J", "", "rbs + crtE in pSB1C3", ""],<br />
["pC13K", "pC13K", "", "crtI in pSB1C3", ""],<br />
["pC13L", "pC13L", "", "rbs + crtI in pSB1C3", ""],<br />
["pC13M", "pC13M", "", "crtB in pSB1C3", ""],<br />
["pC13N", "pC13N", "", "rbs + crtB in pSB1C3", ""],<br />
["pC13O", "pC13O", "", "rbs + crtY in pSB1C3", ""],<br />
["pC13P", "pC13P", "", "trpC promoter in pSB1C3", ""],<br />
["pC13Q", "pC13Q", "", "trpC terminator in pSB1C3", ""],<br />
["pCg13R", "pCg13R", "", "nptII in pSB1C3", ""],<br />
["pCg13S", "pCg13S", "", "nptII biobrick in pSB1C3", ""],<br />
["pAb13T", "pAb13T", "", "PtrpC + bar + Ttrpc + PgpdA + lox in pBARGPE1", ""],<br />
["pCg13U", "pCg13U", "", "nptII biobrick + TtrpC in pSB1C3", ""],<br />
["pCh13V", "pCh13V", "", "hph in pSB1C3", ""],<br />
["pCh13W", "pCh13W", "", "hph biobrick in pSB1C3", ""],<br />
["pCp13X", "pCp13X", "", "bar in pSB1C3", ""],<br />
["pCg13Y", "pCg13Y", "", "PtrpC + nptII biobrick in pSB1C3", ""],<br />
["pC13Z", "pC13Z", "", "GFP + LVA in pSB1C3", ""]<br />
<br />
];<br />
<br />
var parents = document.getElementsByClassName("entry");<br />
for(var pi = 0; pi < parents.length; pi++)<br />
{<br />
var eles = [parents[pi].children[0].children[1], parents[pi].children[0].children[2]];<br />
for(var ti = 0; ti < 2; ti++)<br />
{<br />
var ele = eles[ti];<br />
var text = ele.innerHTML;<br />
for(var i = 0; i < info.length; i++)<br />
{<br />
var p = info[i];<br />
var name = p[0];<br />
var full = p[1];<br />
var img = p[2];<br />
var desc = p[3];<br />
var href = p[4];<br />
<br />
var content = '';<br />
var after = text;<br />
var pos = after.toLowerCase().indexOf(p[0].toLowerCase());<br />
while(pos != -1)<br />
{<br />
var before = after.substring(0, pos);<br />
var actualName = after.substring(pos, pos + name.length);<br />
var middle = actualName;<br />
after = after.substring(pos + name.length);<br />
if( ( before.length == 0 || before.match(/[^a-zA-Z]$/) ) &&<br />
( after.length == 0 || after.match(/^[^a-zA-Z]/) ) &&<br />
(name != 'Eric' || !after.match(/^ Appel/) ) )<br />
{<br />
var image = img ? '<img src="' + img + '">' : '';<br />
middle = '<div class="author-hover"><a href="' + href + '">' + actualName + '</a></div><div class="author-hover-pic"><div class="author-desc">' + image + '<div><h5>' + full + '</h5><p>' + desc + '</p></div></div></div>';<br />
}<br />
content = content + before + middle;<br />
pos = after.toLowerCase().indexOf(p[0].toLowerCase());<br />
}<br />
text = content ? content + after : text;<br />
}<br />
ele.innerHTML = text;<br />
}<br />
}<br />
// <br />
// var hovers = document.getElementsByClassName("author-hover");<br />
// var hoverpics = document.getElementsByClassName("author-hover-pic");<br />
// for(var i = 0; i < hovers.length; i++)<br />
// {<br />
// alert(hovers[i].offsetWidth);<br />
// hoverpics[i].style.marginLeft = 0 - hovers[i].offsetWidth;<br />
// }<br />
}<br />
<br />
function isDelimiter(ch)<br />
{<br />
return ch == ' ' || ch == '.';<br />
}</div>R.Lizarralde