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 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.
-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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{{{tech}}}

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 4th
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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.

The previous days ligations were all desalted and transformed, yet again.
-Kyle

July 5th
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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.

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?
-Kyle

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

July 6th
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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.

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!
-Kyle

July 7th
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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.

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.
-Kyle

Week 4

(07/08 - 07/14)

July 8th
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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.

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.
-Kyle

July 9th
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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!
-Kyle

July 10th
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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.

The nptII ligation with TtrpC grew colonies was unsuccessful. In other news, much lab organization was done today!
-Kyle

July 10th
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{{{tech}}}

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 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.

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.
-Rafael

July 12th
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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.

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

July 13th
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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. 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 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.

Cloning of nptIIBB in front of PtrpC was unsuccessful. Plasmids 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 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.
-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. 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).

Gibson hph was submitted for sequencing, and ALL of the cloning was performed.
-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 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.

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.
-Danielle

July 17th
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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.

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.
-Danielle

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 19th
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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.

Sent pC13AH in for sequencing, and checked several plasmids for the presence of the correct genes using digestion screens and gel analysis.
-Rafael

July 20th
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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.

Plasmid pC13AY was prepared for sequencing, and several transformations using heat shock were performed.
-Rafael

July 21st
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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.

Made cultures of the previous days transformants, and ran an inconclusive digest screen of pAK13AN and pAK13AD. A slow day.
-Rafael

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 22nd
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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.

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.
-Rafael

July 23rd
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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. G. lucidum<i> finished drying.</div>

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!
-Rafael </div> </div>

July 23rd
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{{{tech}}}

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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{{{tech}}}

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

July 24th
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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.

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.
-Rafael

July 25th
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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.

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!
-Rafael

July 26th
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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.

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”.
-Rafael

July 27th
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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.

We retried the PCR of pC13F, ran 63 colony PCRs, and made 97 cultures!
-Rafael

July 28th
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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.

We digested pC13F and miniprepped cultures of yesterday's successful colony PCRs.
-Rafael

July 28th
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{{{tech}}}

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 29th
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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>.

Reran many PCRs and other procedures.
-Rafael

July 30th
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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!

Yesterdays transformations all failed, and other mishaps. But fungal DNA extraction was successful!
-Rafael

July 31st
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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.

Successful returns from sequencing of pC13BD, pCg13Y, pAK13AN and pC13V PstI mutant. In other news, the PCRs of <i>Ganoderma</i> DNA looked good!!
-Rafael

July 31st
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{{{tech}}}

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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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.

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.
-Tina

August 1st
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{{{tech}}}

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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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.

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.
-Tina

August 2nd
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{{{tech}}}

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

August 3rd
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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.

We continued cloning of T7 polymerase behind a constitutive promoter, transforming the GPD homology regions, and biobricking the carotenoid and geneticin resistance genes.
-Tina

August 4th
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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. We transformed and plated pC13M + pAK13D, pCg13S +pAK13D, and pC13K +pAK13D. More LB agar and ampicillin plates were also made today.

We grew cultures of the GPD homology region transformations and transformed yesterday's carotenoid genes and geneticin resistance constructs.
-Tina

August 4th
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{{{tech}}}

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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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.

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.
-Rafael

August 5th
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{{{tech}}}

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

August 6th
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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.

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.
-Rafael

August 7th
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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.

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.
-Rafael

August 7th
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{{{tech}}}

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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{{{tech}}}

The sensor has been connected!
-Rebecca

August 8th
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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.

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.
-Jonlin

August 9th
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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.

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.
-Rafael

August 10th
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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.

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.
-Rafael

August 10th
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{{{tech}}}

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

August 11th
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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.

We are starting cloning of limonene synthase and continuing cloning of the Cre gene. Cloning of fluorescent constructs is going well.
-Rafael

Week 9

(08/12 - 08/18)

August 12th
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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.

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.
-Rafael

August 13th
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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.

We continued cloning of T7 with mRFP, PtrpC with GFP, and PtrpC with mRFP.
-Rafael

August 14th
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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.

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>.
-Rafael

August 15th
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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.

We ran a Gibson assembly to create pC13BH and transformed the construct.
-Rafael

August 15th
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{{{tech}}}

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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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.

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.
-Rafael

August 17th
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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.

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.
-Rafael

August 18th
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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.

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!
-Rafael

August 18
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Today, the dastardly trio of Arun, Hannah, and Eric were chosen to present at the regional iGEM competition!
-Swati

Week 10

(08/19 - 08/25)

August 19th
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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.

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!
-Rafael

August 20th
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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.

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.
-Rafael

August 21st
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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!

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.
-Rafael

August 22nd
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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.

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.
-Rafael

August 23rd
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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.

Cloning of RBS + GFP, RBS + mRFP, PT7 + GFP + PtrpC + geneticin resistance, PtrpC + mRFP, and PtrpC + GFP appear to be going well.
-Rafael

August 24th
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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.

Running into some problems with PgpdA cloning. PCR confirmed that pC13AG and pAK13CR worked! We picked more colonies of pAK13CS for screening.
-Rafael

August 25th
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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.

The extra colonies of pAK13CS from yesterday failed. We will have to restart pAK13CS. We are also recreating inserts from pAK13AB to make pC13AF.
-Rafael

August 25th
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{{{tech}}}

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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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.

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.
-Rafael

August 26th
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{{{tech}}}

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 27th
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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.

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.
-Rafael

August 28th
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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.

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!
-Rafael

August 29th
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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.

Transformed our T7 polymerase ligations. We ligated pAK13CS and pC13CV. We electroporated and plated pC13I, pC13J, pAK13CS and pC13CV -- pC13J unfortunately arced…
-Rafael

August 30th
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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.

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.
-Rafael

August 31st
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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.

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!
-Rafael

September 1st
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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…

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>.
-Rafael

September 1st
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{{{tech}}}

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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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.

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.
-Rafael

September 3rd
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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.

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.
-Rafael

September 3rd
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{{{tech}}}

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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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.

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!
-Rafael

September 5th
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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.

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.
-Rafael

September 6th
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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.

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!
-Rafael

September 6th
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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.
-Arun

September 7th
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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.

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.
-Rafael

September 7th
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{{{tech}}}

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 7th
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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.
-Arun

September 8th
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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.
-Eric, Arun

September 8th
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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.

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.
-Rafael

Week 13

(09/09 - 09/15)

September 9th
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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.

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.
-Rafael

September 9th
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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.
-Eric

September 10th
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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.

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.
-Rafael

September 10th
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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.
-Arun, Eric

September 11th
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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).

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>.
-Rafael

September 12th
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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.

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.
-Rafael

September 13th
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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.

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.
-Rafael

September 13th
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{{{tech}}}

Managed to get the arduino to control the heating circuit!
-Rebecca

September 14th
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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.

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.
-Rafael

September 15th
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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.
-Arun, Eric

September 15th
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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.

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.
-Rafael

Week 14

(09/16 - 09/22)

September 16th
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{{{tech}}}

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.
-Mac


September 16th
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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.

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.
-Rafael

September 17th
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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.

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.
-Rafael

September 17th
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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!
-Arun, Eric

September 18th
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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.

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 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.

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.
-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 prepared and autoclaved CYM + agar, for making plates.

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 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.

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.
-Rafael

September 21st
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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.
-Arun , Eric

September 22nd
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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.

We plated pC13AI. We are continuing <i>Aspergillus</i> growth assay, and completed the <i>G. lucidum</i> growth assay.
-Rafael

Week 15

(09/23 - 09/29)


September 23rd
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{{{tech}}}

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.
-Mac


September 23rd
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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.

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>.
-Rafael

September 24th
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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.

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.
-Rafael

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 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.

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.
-Rafael

September 25th
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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.
-Arun, Eric

September 26th
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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.

The T7 promoter and hygromycin resistance construct appears functional in <i>E. coli</i>.
-Rafael

September 26th
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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.
-Eric

September 27th
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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.
-Arun,Eric

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