Team:Cornell/notebook

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Cornell University Genetically Engineered Machines

Notebook

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January

January 5th
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Applications for new Cornell iGEM members were due today-- we've got 57!
-Rafael

January 16th
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After an eternity of reading and a gigantic spreadsheet of evaluations, we're finally ready to start interviewing applicants-- 36 of them!
-Rafael

January 25th
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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.
-Rafael

January 27th
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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!
-Rafael

February

February 1st
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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!

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.
-Rafael

February 7th
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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.
-Rafael

February 9th
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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.
-Rafael

February 16th
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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.
-Rafael

February 21th
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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...
-Nupur

February 23th
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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 :(
-Rafael

February 28th
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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.
-Nupur

March

March 1st
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Check out our notebook before anything worked!

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.
-Nupur

March 2nd
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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.
-Rafael

March 5th
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We just finished the javascript for filtering. Rafael also made cool subteam icons for the filter buttons.
-Nupur

March 6th
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I made a template for notebook entries and added styling to each entry.
-Nupur

March 9th
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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!
-Rafael

March 29rd
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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.

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.
-Rafael

April

April 6th
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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.
-Rafael

April 10th
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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!
-Rafael

April 13th
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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!
-Rafael

April 20th
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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.
-Rafael

April 26th
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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.
-Rafael

May

May 4th
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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!
-Rafael

Week 1

(06/17 - 06/23)

June 17th
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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.
-Mac

June 18th
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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).

We started bootcamp today, doing our first transformations with kit plate biobricks.
-Rafael

June 19th
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From the plasmid pUCATPH (pAh13G), we amplified the trpC promoter (PtrpC, a constitutive fungal promoter) and the trpC terminator (TtrpC). From pNG (pAg13H), we amplified nptII, the geneticin (a plant/fungal antibiotic) resistance gene.

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.
-Rafael

June 20th
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Check out the gif 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.

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.
-Nupur

June 20th
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We miniprepped the crtEIB (BBa_K523022), crtY (BBa_K539119), and T7 (BBa_I712074) biobricks. The gels confirmed our PCRs for PtrpC, TtrpC, and nptII, which we then digested to insert into pSB1C3.

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.
-Rafael

June 21st
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The javascript for toggling technical details took a bit longer than expected, but we finished it up today.
-Nupur

June 21st
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PtrpC, TtrpC, and nptII were column-purified, digested pSB1C3 backbone was gel-purified and dephosphorylated, and each was ligated into the pSB1C3 backbone.

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.
-Rafael

June 22nd
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I added a flyout to notebook entries authors with a picture, short description, and link to their bio on the team page.
-Nupur

June 22nd
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We transformed TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R) constructs into DH5α via electroporation.

Our penultimate day of bootcamp was a light one-- we only transformed cells with the constructs we'd made over the past four days.
-Rafael

June 23rd
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We used Q5 PCRs with the standard biobrick primers VF2 and VR to attempt to confirm the presence of TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R).

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.
-Rafael

Week 2

(06/24 - 06/30)

June 24th
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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.
-Mac

June 24th
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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.
-Nupur

June 24th
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Minipreps of TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R) were performed with an EZNA kit, and afterwards we quantified with a nanodrop.

We miniprepped our three constructs. Then we went back to sleep.
-Rafael

June 25th
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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.
-Nupur

June 25th
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The Q5 PCRs of TtrpC (pC13Q), PtrpC (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.

We did another PCR from the miniprepped plasmids to confirm the right sizes of inserts, then sent them in for sequencing.
-Rafael

June 26th
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{{{tech}}}

The script is now working on all browsers except for Internet Explorer.
-Nupur

June 27th
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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).

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.
-Rafael

June 28th
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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).

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.
-Rafael

June 29th
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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.

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.
-Rafael

June 30th
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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.

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.
-Rafael

Week 3

(07/01 - 07/07)

July 1st
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Now we just have to start filling out entries...

We finished the hypertexting script and fixed some miscellaneous problems. I think we can call this a wrap. The notebook page is officially done!
-Nupur

July 1st
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Price of one 12 oz Styrofoam cup: 4.09 cents

Two of our team members participated in SILS Skills Night on June 20th.
-Hannah

July 1st
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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.

Yesterday's digestions were gel extracted, ligated, and transformed. Hopefully it worked this time!
-Rafael

July 2nd
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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.

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.
-Rafael

July 3rd
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:( ;_; 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>

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.
-Rafael

July 5th
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{{{tech}}}

Rafael added badges to the headers of all of our wikis, and implemented a badge flyout that links them all together.
-Nupur

Week 4

(07/08 - 07/14)

July 10th
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Five team members visited a STEM camp in Canandaigua, NY. We spent two hours making DNA necklaces with kids ranging from ages 7-14.
-Hannah

July 11th
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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.
-Rebecca

July 11th
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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.

Digest screens were run to see if cloning had been successful,, and Gibson cloning was continued.
-Rafael

July 12th
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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.

Cloning with nptII BB and PtrpC was continued, as well as cloning with the Gibson Assembly method.
-Rafael

July 13th
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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.

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.
-Rafael

July 14th
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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.

Our cloning of pCg13Y was found to be unsuccessful. pC13z and pAK13AD were prepped for cloning.
-Rafael

July 14th
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{{{tech}}}

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.
-Mac

Week 5

(07/15 - 07/21)

July 15th
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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.
-Rebecca

July 15th
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Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. Gibson hph was submitted for sequencing, and pAK13ADs 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).

Gibson hph was submitted for sequencing, and cloning of crtY and crtB was continued.
-Rafael

July 16th
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Price of one 12 oz Styrofoam cup: 4.95 cents

One iGEM member spoke at a panel for pre-freshmen biology students interested in pursuing research positions in college (PSP).
-Hannah

July 16th
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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.


-Rafael

July 18th
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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.

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.
-Danielle

July 21st
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{{{tech}}}

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.
-Mac

Week 6

(07/22 - 07/28)

July 23rd
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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.
-Rebecca

July 23rd
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Five team members gave a two hour presentation including a lab tour to a group of 25 high school biology teachers (CIBT)
-Hannah

July 28th
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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.
-Mac

Week 7

(07/29 - 08/04)

July 31st
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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.
-Rebecca

August 1st
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Team visited Ecovative Design's facilities in Troy, NY. Co-founder and Chief Scientist gave us a tour and tips on working with fungi.
-Hannah

August 2nd
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Video conference with Tuft's iGEM team to discuss collaboration on their human practices approach.
-Hannah

August 4th
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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.
-Mac

Week 8

(08/05 - 08/11)

August 5th
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Eric Holmes and Arun Chakravorty presented for a group of scientific researchers (SILS)
-Hannah

August 7th
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We helped 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!
-Nupur

August 7th
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The sensor has been connected!
-Rebecca

August 10th
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Five team members presented at the Ithaca Sustainability Center for citizens of Tompkins County.
-Hannah


Week 9

(08/12 - 08/18)

August 14th
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Carotenoids
Alex: Made more LB. Still no mini-prep kit aauughhh…
-Danielle

August 15th
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Carotenoids
Alex: Made LB stocks of A D F K L M N AC?x3 BB?x3
-Danielle

August 15th
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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.
-Rebecca

August 16th
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Carotenoids
Alex: Actual mini-prep kit arrives!!!
Alex: ACx3 LB stock failed, wrong antibiotic
Alex: Mini-prepped A D F K L M N BBx3 with decent yields for most. BBx3 not good enough for sequencing...
Alex: Remade ACx3 (new colonies) LB stocks
Alex: Plated A D F K L.
Alex: Turns out standard biobrick primers may not have been used for previous (Aug. 5) colony PCRs (AB? AC? BB?).
Should Colony PCR these tomorrow from LB stocks/mini-preps. Remaking ABx3 LB stocks as well.
Alex: Still need to test whether rbs + crtE can be extracted. Run PCR of this with the others tomorrow.
Alex: Ran PCR of AB? with primers 23 & 24.
-Danielle

August 17th
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Carotenoids
Alex: Mini-prepped redone ACx3.
Alex: PCR: ACx3, BBx3, A (rbs + crtE, crtE).
Alex: Gel: ABx3, ACx3, BBx3, some of them worked with each. rbs + crtE, crtE failed (why?).
Alex: Remade larger stocks of ABx3, ACx3, BBx3; apparently, 5mL cultures aren't good enough for sequencing. Also, stock of A, quality was poor.
Alex: PCR--Retried rbs + crtE and crtE from A, with good quantity of mini-prep, using Q5.
-Danielle

August 18th
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Carotenoids
Alex: Mini-prepped 10mL ABx3, ACx3, BBx3, A. Will nanodrop tomorrow. Plated cultures as well.
Alex: Ran PCR of the above to verify, and to test again for inserts for I and J.
-Danielle

Week 10

(08/19 - 08/25)

August 19th
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Carotenoids
Alex: Forgot to put in plates last night... Put them in at 11:00am today. Take out during evening please.
Alex: Nanodrops were okay for most samples... AB_c, which was spilled, was not very good. A is bad again (what is it not working???)
Alex: Running gel of PCRs from Aug. 18 and Aug. 17. GREAT SUCCESS!!! AB all good, AC all good, BB all good, A (old and new, I and J inserts) all good! (Don't forget to Q5 2step next time for I and J).
Rebecca: 1step Q5 PCR of minipreps of N (rbs+crtB) & L (rbs+crtI)
Alex and Rebecca: Made 500ml LB & 2X LB+agar (250ml). Made 23 plates (no antibiotic).
Alex and Rebecca: Ran agarose gel of PCRs of minipreps of N&L. Gel ran well.
-Danielle

August 20th
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Carotenoids
Rebecca: PCR clean up of AB,AC,I,J,N,L
Alex and Rebecca: Double digests of inserts and vectors for I, J, AF, AG, CR, CS overnight
-Danielle

August 21st
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Carotenoids
Alex: Digest cleanup and quantification. A2944(X,P) had no DNA. F(X,P) wouldn't come out of the column… Redo both. Everything else okay.
Alex: Ligation of AF, AG, CR and CS did not work well—forgot to dephosphorylate. May still have acceptable yields, but we'll see.
Alex: Dephosphorylating backbones for a redo
Alex: Electroporation of AF, AG, CR, CS good only. Not enough cuvettes for the rest.
Alex: Plated AF? AG? CR? CS? for growth overnight.
-Danielle

August 22nd
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Carotenoids
Rebecca: At 9am, there was nothing on the plates AF? AG? CR? CS?
Rebecca: PCR clean up of A(30,44) & A(29,44) for I & J. Double digested with restriction enzymes X & P at 11.35am. Incubate at 37C for 1h 30mins. Heat kill in water bath for 20mins. Clean up and stored in the wells nearest to opening of the freezer on the green rack on the left side of -20C (on top of bench, I took it out from the one below as that's somewhere between 0 to 10C only)
Rebecca: Double digested F with restriction enzymes X&P. Incubate at 37C at 1:05pm
Alex: AG, CR, CS have colonies now. AF doesn't, probably because it arced during electroporation.
Alex: Heat killed digested F. Dephosphorylated.
Alex: Running ligation overnight for I, J
Alex: Created 10mL cultures of AG?, CR?, CS?.
-Danielle

August 23rd
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Carotenoids
Alex: Plated AG?, CR?, CS?
Alex: Mini-prepped AG?, CR?, CS?
-Danielle

August 24th
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Carotenoids
Rebecca: Dilutions for PCR (Took 1 ul of miniprep + 99ul H20, & take 1ul of dilution for PCR template). Run Q5 PCR of AG? (PT7+crtI), CR? (PT7 + rbs + crtB), CS? (PT7 + rbs + crtI) using biobrick primers 23 and 24. [CrtI (~1.5kb), CrtB (~1kb)]
Rebecca: Ran a 1% agarose gel to check band size. AG 1,2 and CR 1 worked. The rest (AG3, CR2, CR3, CS 1,2,3) didn't show anything.
Alex: Picked out 5 more colonies from CS. Also replated AG1, because tripartite plate was getting messy (it was probably wet).
-Danielle

August 25th
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Carotenoids
Alex & Sara: Mini-prepped and plated CS?4-8
Alex & Sara: run PCR for CS?4-8
Sara: Gel showed CS? all failed…
Sara: Running Q5 of AB, for recreating AF.
-Danielle

August 25th
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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.
-Rebecca

Week 11

(08/26 - 09/01)

August 26th
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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.
-Mac

August 26th
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Carotenoids
Sara: Column purifying insert from AB
Alex: Running Q5 of L, for recreating CS
Alex and Danielle: Ligation of AF (insert AB and vector F) using E & P restriction enzymes
Rebecca and Danielle: PCR cleanup of PCR of L (rbs +crtI), double digest. Incubated at 37C at 5:45pm. Heat kill at 80C at 7pm.
-Danielle

August 27th
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Carotenoids
Rebecca and Danielle: Set up PCR for CR1 at 10.20am (estimated done time noon)
Rebecca: PCR clean up of CR1
Rebecca: Double digest of CR1 with X&P + heat kill + clean up. Stored on green rack in fridge.
Rebecca and Danielle: Clean up of insert L(rbs+crtI) for CS (rbs+crtI+T7). Stored on green rack in fridge.
Rebecca: Submitted AG1 and AG2 for sequencing [crtI, T7, pSB1C3]
-Danielle

August 28th
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Carotenoids
Alex: We're definitely out of mini-prep F. I suspect another group may have used ours, since everyone needs it…… I'm going to make a large batch of F LB stock, as well as D. I can mini-prep it tomorrow morning and digest as well. No desalting membranes yet.
Alex: AG1 and AG2 both good! Hooray!
-Danielle

August 29th
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Carotenoids
Alex: 4 Mini-preps of F, 2 mini-preps of D.
Alex and Sara: Ligation CV and ligation CS.
Sara: Digest vec. D, S & P, vec. F, E & P.
Alex: LB stock of AB and AG for glyc. stock.
Alex & Tina: Electrophoretionà Only I, AF, and CV worked. All J lost. Three working ones are plated.
Tina: Q5 2 step for insert for recreating J.
-Danielle

August 30th
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Carotenoids
Alex: Q5 failed. Need to redo.
Sara: Made glyc. stocks of AB and AG.
Alex & Danielle: Attempting to Amplify J insert with Phusion (there's VERY LITTLE Phusion left, maybe like 1uL? Need to spin the tube down first to get it. PCR tube stored in 4C after it's done → Need to run a gel)
Tina: made overnight cultures of I (5 cultures), AF (5 cultures), CV (4 cultures, pretty low # colonies and CV4 may be a mixed colony) ligations
-Danielle

August 31st
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Carotenoids
Tina + Rebecca: Made glyercol stocks of overnight cultures of I, AF, CV in case we have cells if one works (stored in the Corning box in the -80C to the left of the shelves).
Tina, Eric, Rebecca: Electroporation for leftover CS [rbs + crtI (kit) in PSB1AK8 + T7 (D) Amp + Kan] ligation, plated on Am and stored at 37C overnight
Rebecca: Miniprepped overnight cultures of AF1,2,3,4,5 [crtB in PSB1C3 + T7 (F) Cm], CV1,2,3,4 [crtB +rbs in PSB1C3 + T7 (F) Cm] & I1,2,3,4,5 [crtE + PSB1C3 (F)]. Stored in green rack in -20C.
Sara & Alex: Running gel to confirm J
Tina: Digest (for screening) for mini-prep cultures I AF CV and heat killed
Alex: PCR of J worked! (although the band is faint)
-Danielle

September 1st
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Carotenoids
Danielle & Sara: PCR clean up of A(29,44) → need to quantify with Nanodrop; stored in 4C
Sara: Ran digest screens of I, AF, CV-only one (AF2) appeared to work… (Alex, confirm image 9_01_carrot!)
Alex: quantified A(29,44) with Nanodrop
Alex & Danielle: set up PCR Vent reactions for: AF 2, 3 & 4; CV 2, 3, & 4; I 2, 3 & 4; mini L. All failed.
Danielle: dilution of primers 23 and 24 (100uL prepared)
-Danielle

September 1st
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Lydia's back too! Discussed what we have been doing for the past few weeks and immediate goals.
-Rebecca

Week 12

(09/02 - 09/08)

September 2nd
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Carotenoids
Tina & Danielle: Run gel of A2944 (50min run time at 100V, incubation in EtBr for 30min) → PCR failed (no band; ladder looks good)
Danielle: redid PCR with the addition of MgSO4 for AF 2, 3 & 4; CV 2, 3, & 4; I 2, 3 & 4; mini L (10 samples. PCR protocol: iGEM Vent. Estimated end time 7:40pm).
Alex: made plates for CHL LB plates and CYM (fungal media) plates for others
Alex: Dephosphorylating vectors… we didn't do this earlier?!?
-Danielle

September 3rd
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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.
-Rebecca

September 4th
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Carotenoids
Alex: Q5 arrived! PCR and gel of AF, CV, I, 2-4 and ins. L. All failed… except AF2. What's going on???
Alex: PCR ins. A2944. Failed.
-Danielle

September 5th
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Carotenoids
Tina: Made 1 ng/ul dilutions of CV1 and I1.
Tina: Colony PCR of CV1 and I1. Use Q5. Use Primers 23 and 24. In same run, create insert L using Alex's 1:100 dilution. Left overnight in PCR machine.
-Danielle

September 6th
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Carotenoids
Sara: Submitted AF2 for sequencing.
Alex: Made 3 more LB stocks of colonies of CV and I (6-8).
Alex: Yesterday's Q5, CV1, I1, L failed… Eric and I suspect something is definitely wrong with our reagents. New dNTPs have arrived, TRY USING THESE.
-Danielle

September 7th
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Carotenoids
Danielle, Sara, Alex & Rebecca: redo Q5 PCR for CV2-4, I2-4, AF1,3,4 , ins. L with new dNTPs
Alex, Sara, Danielle: Spun down CV6-8 and I6-8. Will mini-prep if above PCRs fail.
Alex, Sara, Danielle: Made LB stocks of AG and AH for biobrick submission
Alex: All AF, CV, I bad. L okay. Suspecting that electroporation did not go well to begin with.
Alex: Running Q5 2 step ins. A2944.
-Danielle

September 7th
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Five team members went to the Ithaca Sciencenter and taught a group of 18 children and 17 parents about the power of DNA.
-Hannah

September 8th
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Carotenoids
Rebecca & Sara: Ran a gel of A2944. Couldn't get the camera to be connected to the computer but took an image just using the camera. It seems that the band is around 2.5kb? Sara told me that it should be about 1kb?
Danielle: got the camera connected again! Transferred Rebecca's image to the computer.
Rebecca: PCR clean up of A2944 [rbs+crtE] for J[rbs+crtE in pSB1C3(F) Cm] and insert for L [rbs+crtI in pSB1C3(F) Cm]
Sara & Rebecca: Digest F with X,P , heat killed. Stored in fridge.
Rebecca: Miniprepped CV6-8 [rbs+crtB in PSB1C3 + T7(F) Cm], I6-8 [crtE in PSB1C3(F) Cm], AH [crtY in PSB1C3+T7(F)], AG [crtI in PSB1C3+T7(F) Cm]. Stored in fridge.
-Danielle

Week 13

(09/09 - 09/15)

September 13th
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Managed to get the arduino to control the heating circuit!
-Rebecca

Week 14

(09/16 - 09/22)

September 16th
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We had no successful fungal transformants of pC13BR, pC13CD, pUCATPH, and pC13AY. We started taking measurements of Ganoderma growth on CYM plates (plated on September 9) 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.

We started taking measurements of Ganoderma 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 pC13K. We still do not have any growth on antifungal plates.
-Rafael

September 17th
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We attempted transforming Cochliobolus 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 chloromphenicol 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.

We tried transformation of Cochliobolus 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.
-Rafael

September 18th
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We used less hygromycin for transformation because Cochliobolus concentrations are less than Ganoderma concentrations. We are recording more and more data with regards with fungal resistance! We found a new contaminant on one plate but we will keep an eye out for it and make sure it does not come into contact with Ganoderma. 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.

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.
-Rafael

September 19th
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We quantified the holin minipreps. Both were around 48ng/uL, which is 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.

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.
-Rafael

September 20th
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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 will start a new growth assay to test afp1. We ran a Q5 PCR for pAK13CR.
-Rafael

September 21st
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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 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.
-Rafael

September 22nd
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We did a PCR cleanup of the pC13AI Gibson pieces, electroporated (time constant was 2.88 - it arced), and plated the cells, We continued taking masses for Aspergillus with afp1 growth assay. We began growth assay with inoculating discs.

We plated pC13AI. We are continuing the Aspergillus growth assay.
-Rafael

Week 15

(09/23 - 09/29)

September 23rd
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Two gels, each with 20 wells, were run simultaneously. 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 E. coli. A culture of G. lucidum 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 re-cultured (with IPTG) pC13BA. The pC13BN transformations yielded no colonies.

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 E. coli for characterization, and set up a zone of inhibition experiment to test the efficacy of antibiotics and antifungals on G. lucidum.
-Danielle

September 24th
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We miniprepped the pC13AI cultures. We amplified with 45/52 and 48/51 and ran a gel, and got products that are 1.5kb. Nothing appeared on the gel, not even the ladder. The EtBr needs to be replaced.

The gel of pC13AI showed nothing - the EtBr (for gel staining) needs to be replaced.
-Danielle

September 25th
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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 continue to take measurements for growth assay. We put a lot of aspergillus around the inoculating discs for gathering more data.

Sequencing of pC13CO did not turn out to be positive. Still trying to get data from aspergillus and growth assay.
-Danielle