Team:Cornell/notebook

From 2013.igem.org

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<h5 class="centered">(09/02 - 09/08)</h5>
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|head=September 2nd
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|text=Miniprepped PtrpC + ''T7 polymerase'', the ''PpelA'', the ''Anderson promoter'', and the ''Anderson promoter'' + ''T7 polymerase''. We are preparing to send pC13BN to sequencing. A Vent PCR was done on pC13CB to verify successful cloning. We began a new transformation attempt, now with fresh solutions to work with. However, this also seemed unsuccessful. We also ran a Vent PCR with the addition of MgSO4 for pC13AF, pC13CV, pC13I and pC13L minipreps--they all failed. Another Vent PCR was run to check inserts in pC13BC, pC13BV, and pC13CE, but resulted in no bands.
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|tech= We got colonies for pAK13CK, ran 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.
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|author=Rafael
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|head=September 3rd
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|text= Digest screen of PtrpC + ''T7 polymerase'' and the ''Anderson promoter'' + ''T7 polymerase'' showed good expected banding pattern. The pC13CK construct is correct. A gel of pC13CB PCR products was run and was inconclusive. We repeated the PCR from yesterday for pC13AF, pC13CV, pC13I and pC13L but using the Q5 polymerase. Except for pC13AF from colony #2, all else failed. We attempted to protoplast again, this time with emphasis on determining what our issue is in hopes that we can fix it.
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|tech= We quantified digestions and ligated pC13BQ + pC13BN, pC13Y + pC13BN, pC13BR + pC13BN, and pC13CD + pC13BN. We electroporated the ligations. We miniprepped pC13CK cultures and ran digest screen, which was successful. The construct is correct. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin'', which was unsuccessful. The pC13AY, pA13AM, and pA13AW preps were digested with EcoRI and PstI and run on a gel. Results indicate that pC13AY and some of the pA13AW colonies worked. The gel of Vent PCR of pC13CB did not turn out too well. We got a bunch of lanes with smeared rather than distinct bands. We then ligated and transformed ''T7 promoter'' with ''mRFP'' to obtain pAK13AO and ''PgpdA'' with ''GFP'' to create pC13CF. We ran a Q5 PCR and gel for pC13AF, pC13CV, pC13I and pC13L. Sadly, they all failed except pC13AF from colony #2. We are concerned with what’s going on. The PCR for pC13A failed again too. We miniprepped our pAh13G cultures. Our new fungal culture doesn’t have nearly as much mass in it, leading us to believe that the old cultures were almost definitely contaminated with bacteria. Our transformed pC13BK and pC13BM grew into masses covering the plates rather than colonies, and the others didn’t grow. We restreaked pC13BK and pC13BM hoping for single colonies. We attempted a diagnostic protoplasting, determining that pellets formed are likely from contaminations, as the new cultures with ampicillin did not produce pellets. We also tried to manually extract mycelium and enzyme digest it, but microscope visualization did not show signs of protoplasting having occurred. Even after filtering through one layer of cheesecloth and nylon, there was no appearance of protoplasts.
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|author=Rafael
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|head=September 4th
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|text= Transformed the ''PpelA'' and ''Anderson promoter'' + ''T7 polymerase'' into pSB1C3 as well as digest and ligate other promoters. The pC13BN construct was incorrect, so we will have to redo all of the Gibson transformations for the ''lox'' constructs. On a brighter note, sequence confirmation of pA13DB and pA13DC came back today!
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|tech= We got transformants for all ''lox'' constructs. We got sequencing back for pC13BN and it was incorrect, so we will redo the Gibson transformation and all the subsequent ''lox'' constructs. We also electroporated pC13BF and pC13AZ and digested pC13F and pA13AW with XbaI and PstI for ligation to make pC13AZ. pC13F and pC13BE KpnI and BamHI digests were then ligated to make pC13BH. pA13DB colony 1 and pA13DC colony 4 were sequence confirmed today. Lastly, we made cultures from restreaked pC13BM and pC13BK plates.
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|author=Rafael
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|head=September 5th
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|text= Today in lab we gel screened a bunch of old minipreps to see if they were still viable and made cultures of the ''Anderson promoter'' + ''T7 polymerase'' in pSB1C3. We digested the ''lox'' site constructs in order to ligate with pC13BN. In a meeting with Professor Turgeon we decided that we would leave <i>G. lucidum</i> behind for now and work with <i>Cochliobolus</i>, which her lab transforms regularly and has more expertise with.
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|tech= The Gibson was digested with DpnI and electroporated into DH5a. We are growing DH5a for electrocompetent stocks. We did a PCR of pSB1C3 to append cut sites in order to ligate with ''holin''. We digested pC13Y, pC13BQ, pC13BR, and pC13CN for ligation with pC13BN and made cultures of three pC13AZ colonies. Gel screen of pC13F and pA13AW appeared successful and were then column purified. We also gel screened pC13P miniprep, SpeI and PstI digested pC13P, PCR of ''PgpdA'' for homologous regions, PCR of pC13F for homologous regions, and BamHI/KpnI digested pA13BE to verify the viability of old minipreps. We then amplified ''T7 polymerase'' from pC13E. We did a Q5 PCR for pC13CV, pC13I and pC13L. Our plates from transformation on Saturday showed conidia growth on the controls with no hygromycin. None of the plates with hygromycin showed any growth. We decided that we will now use <i>Cochliobolus</i> to protoplast and transform after meeting with Professor Turgeon again. To determine if the DNA is in the fungal genome and stable, we will plate on non-antibiotic and antibiotic plates, then replate transformants from the antibiotic plate onto non-antibiotic plates and back onto antibiotic plates. Since protoplasting is failing, we will turn to agrobacterium for <i>G. lucidum</i> transformation in the future. We will also check for homology between the promoter gpd in <i>G. lucidum</i> and <i>Cochliobolus</i> to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after <i>Cochliobolus</i> gpd by cutting into that region in the genomic promoter to transform. We also created glycerol stocks of pC13BM and pC13BK cultures and miniprepped them to submit for sequencing. We set up new cultures of colonies from transformation of pC13BC, pC13BV, and pC13CE, and ran colony PCRs of them (using our brand new tube of Q5 polymerase!) to check for inserts.
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|author=Rafael
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|head=September 6th
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|text= Most of old minipreps were no good and the ''PpelA'' transformation failed. The ''Anderson promoter+T7 polymerase'' gel results looked good and were submitted for sequencing. Oh no, the PCR of pC13CV, pC13I and pC13L failed again--we suspect something is wrong with our reagents. Use the new dNTPs from now on!
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|tech= We digested, ligated, and transformed to create pC13DD. We ran a PCR to append cut sites to pSB1C3 for digestion and ligation with ''holin''. Gel results indicate that pC13P miniprep, pC13P SpeI/PstI digests, ''PgpdA'' PCR for Gibson and the pA13BE BamHI/KpnI/SacI digest are not viable, but the pC13F PCR for Gibson might be okay. pC13BF transformation failed as well. The pC13AZ cultures were digest screened; results were good, so they were sent for sequencing. pC13P was plated and pC13E was cultured. We submitted pC13AF (colony #2) for sequencing. The Q5 PCR from yesterday--pC13CV, pC13I and pC13L--was a failure. We suspected that something is wrong with our reagents. Use the new dNTPs from now on! We ran PCRs to prepare the necessary parts for our Gibson assembly to produce parts with flanking <i>G. lucidum</i> homologous regions. We spun down the cultures of pC13BC, pC13BV, and pC13CE and stored them in the fridge.
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|author=Rafael
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|author=Arun
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|head=September 7th
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|text= Digested and ligated the ''Anderson promoter+T7 polymerase'' into pSB1C3 and pSB2K3 as well as start cultures from glycerol stocks and transformation and gel screened the ''Anderson promoter'' + ''T7 polymerase''. We transformed ''afp1'' and ''Cht1_2''. For fluorescence, we electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ to obtain more plasmid DNA. We redid a Q5 PCR for pC13CV, pC13I, pC13AF and pC13L with the new dNTPs--PCR products were all bad except for pC13L.
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|tech= We did Q5 PCR to amplify pSB1C3 for Gibson with ''afp1'' and ''Cht1_2''. 3uL of PCR product was run on the gel, found to correct, and was cleaned. We ran a Gibson of pSB1C3 with both pC13CL and pC13BA. Both of these were transformed, ''Cht1_2'' into DH5a (time constant 4.82) and ''afp1'' into BL21 (time constant 4.84). We made glycerol stock of pC13BN. We ran a gel of pSB1C3 PCR from yesterday, which was successful, and performed a PCR clean up. We digested pSB1C3 and pC13CN with HindIII and BglII. We ligated these together to create pC13CO and transformed it into DH5a (time constant 4.82). We miniprepped pC13BN culture and did a double digest on pC13BN culture with KpnI and HindIII to see if internal cut sites were present. There was some digestion that occurred when it was run on a gel, so we are confident the sequence is correct. We double digested pC13BN again with KpnI and HindIII. We ligated it with pC13Y (time constant 4.86), pC13BR (4.86), pC13BQ (4.90), pC13CD (4.80). We got transformants for pC13DD. A colony PCR was run and pC13DD colony 3 looked good. This colony was grown in liquid culture for miniprepping and sequencing. We grew cultures of pC13BX, pC13CK, and pC13BN for submission (pC13BX submitted directly; pC13CK and pC13BN need sequence confirmation first). pC13BF transformants were cultured, pC13AZ cultures were digested with EcoRI/PstI and gel screened; results were good, so they were submitted for sequencing. Digested pK13AL and pA13AW with XbaI/PstI for ligation to make pK13AX. pC13P and pC13E were digested with EcoRI/PstI and ligated to make pC13AY. Cultures of pC13Q and pC13BE were made from glycerol stocks. We electroporated pC13AU, pC13AT, pAK13CY, and pC13AQ. We also ran a Q5 PCR of pC13CB and pAK13AO using standard BioBrick primers 23 and 24 in order to confirm lengths of inserts. We redid the Q5 PCR with the new dNTPs for pC13CV, pC13I, pC13AF  and pC13L--pC13CV, pC13I and pC13AF were bad, but pC13L was good! We suspect that electroporation did not go well to begin with. We reran the 2-step Q5 PCR for pC13A. We ran a gel of our PCR to create Gibson assembly parts, and it appeared unsuccessful. We ran the colony PCRs of pC13BC, pC13BV, and pC13CE on a gel, and only observed bands for a few pC13BC colonies.
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|head=September 8th
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|text= We miniprepped ''PpelA'', PtrpC, and ''TtrpC''. We got transformants with ''afp1'' and ''Cht1_2'' and for all of the ''lox'' site constructs. However, only the pC13CO looked good based on a gel screen. We will sequence the ''lox'' construct. We also miniprepped pC13CV, pC13I, pC13AH and pC13AG.
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|tech= We got transformants from the ''afp1'' and ''Cht1_2'' Gibsons. There were a lot of red colonies and very few white ones, and an attempted pick and streak of the white colonies was performed. We got transformants for pC13CO, pC13BN + pC13CD, pC13BN + pC13Y, pC13BN + pC13BQ, and pC13BN + pC13BR. A colony PCR was performed, and a gel of the PCR products looked good for only pC13CO colony 2. No positive inserts were present, so maybe the ''lox'' construct is not correct. To check, we will sequence it tomorrow. Afterwards, we miniprepped pC13BF, pC13P, and pC13Q and digested pC13BF. We ran the gel for pC13A and did a PCR clean up for it, as well as for pC13J and pC13L. We also digested pSB1C3 with XbaI and PstI, and miniprepped pC13CV, pC13I, pC13AH and pC13AG. We attempted to run another gel of our PCR product to create Gibson assembly parts, which looked even worse than yesterday. We also did a PCR cleanup of pC13BJ, pC13BK, pC13BL, and pC13BM.
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|author=Rafael
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Revision as of 02:11, 28 September 2013

Cornell University Genetically Engineered Machines

Notebook

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January

January 5th
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{{{tech}}}

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

January 16th
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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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

March 6th
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{{{tech}}}

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

March 9th
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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

Today we worked on team structure for the summer; Swati will be the overall team leader, with me and Mark coordinating wetlab, Olya taking meeting minutes, Mac running drylab, and Hannah will be managing human practices. Later on in the summer, we will also develop task groups. For the next week, we've also split into groups to research various aspects of the mycelial materials project.
-Rafael

April 26th
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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

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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{{{tech}}}

I fixed a problem with the hypertext script not looping, but broke something else that causes everything to crash. I'm going to sleep. I'll figure it out tomorrow.
-Nupur

June 25th
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The Q5 PCRs of TtrpC (pC13Q), PtrpC (pC13P), and nptII (pCg13R) showed bands of the correct size, so sequencing with VF2 and VR was submitted to make sure nothing strange had happened.

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

June 26th
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{{{tech}}}

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

June 27th
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pC13B (crtY) was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). However, the gel showed only one band for each (what one would expect for pAK13D, but pC13B should have had two bands).

We started cloning crt genes downsteam of the T7 promoter. Because the T7 promoter is too small to extract as a fragment, we're placing crt genes downstream, then transferring the promoter+gene fragments back to pSB1C3. Today we digested crtY and the T7 promoter plasmid, but the digestion of crtY failed.
-Rafael

June 28th
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pC13B (crtY) was digested with XbaI and PstI, and pAK13D (T7 promoter) with SpeI and PstI (standard RFC 10 cloning). The gel this time had two bands at the correct locations for pC13B, so we're hoping it's a success this time. We also started overnight LB cultures of pAb13T (pBARGPE1).

We redid the digestion of crtY and the T7 promoter plasmid from yesterday, apparently successfully this time. We also started liquid cultures of pBARGPE1, a plasmid we got from the Fungal Genetics Stock Center.
-Rafael

June 29th
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If successful, the ligation will form pAK13AD. pC13K (crtI, BBa_K118003) and pC13M (crtB, BBa_K118002) were digested with XbaI and PstI for insertion into pAK13D, and separated on a gel. pAb13T (pBARGPE1) was miniprepped and quantified by nanodrop.

Crossing our fingers, we gel extracted and ligated the ostensibly successful digests of crtY and the T7 promoter plasmid, and then transformed. We also digested crtI and crtB, other carotenoid genes that we will also be putting under the T7 promoter, and miniprepped the pBARGPE1 that had been grown up last night.
-Rafael

June 30th
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pC13K (crtI, BBa_K118003) and pC13M (crtB, BBa_K118002) were digested with XbaI and PstI, pAK13D with SpeI and PstI, pC13Q (TtrpC) with EcoRI and XbaI, and pCg13R (nptII) with EcoRI and SpeI, and all were separated on a gel; crtI looked faint, and pAK13D was smeared, but all fragments were cut out for purification tomorrow.

Unfortunately, there were no colonies on the transformation plate for T7+crtY, so we redid the digestions for those. We also digested TtrpC and nptII for the eventual cloning of a PtrpC+nptII+TtrpC construct (we have to put the parts together in reverse because nptII contains a PstI site, which we will be trying to remove as well). Gel extractions of digested crtI and crtB were also empty (insignificant concentrations), so these digestions were also redone, and all were run on a gel, which looked mostly okay.
-Rafael

Week 3

(07/01 - 07/07)

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

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

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

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

July 1st
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Digested pAK13D was dephosphorylated, then crtI , crtB, and crtY were ligated with pAK13D to put them under the T7 promoter, hopefully producing pAK13AB, pAK13AC, and pAK13AD, respectively. pCg13R was also ligated with pC13Q, hopefully producing pCg13U. All four constructs were transformed into DH5α via electroporation.

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

July 2nd
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hph was amplified with Gibson primers from pAh13G, and the pSB1C3 backbone was amplified with Gibson primers from pC13F (BBa_J04450). When assembled, they will form pCh13V.

There were no transformants, again :(. On the other hand, we received our tube of Ganoderma lucidum from Dr. David Hibbett today! Once more with feeling, we redid the four digestion-ligations for crtI, crtB, crtY, and nptII. We also amplified hph, the hygromycin resistance gene, from one of the plasmids we got from Dr. Turgeon's lab, as well as pSB1C3 backbone, and we will use Gibson assembly to put them together (as hph has both EcoRI and PstI sites)-- the gels looked good for both of these. Finally, we submitted pAb13T (pBARGPE1) for sequencing to determine the sequences of the Aspergillus nidulans gpdA promoter and the bar gene, which confers phosphonothricin resistance.
-Rafael

July 3rd
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:( ;_; T_T D: (tears for the transformations lost). The bar PCR and pC13F were digested with EcoRI and PstI, and ligated to hopefully form pCp13X. Site-directed mutagenesis was performed both with and without DpnI to digest template DNA. <add something about site-directed mutagenesis design; refer to primers? probably requires Swatinput>

All transformations failed yet again, and were repeated once more. Four tears shed for the lost. hph was Gibson-assembled, and we ran a Q5 PCR of the bar gene, under the assumption it matches other published bar sequences, and the product digested and ligated with pSB1C3. We also ran site-directed mutagenesis on nptII (pCg13R) to remove its internal PstI site.
-Rafael

July 5th
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{{{tech}}}

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

Week 4

(07/08 - 07/14)

July 10th
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{{{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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{{{tech}}}

After several brainstorming sessions, we decided to build an intelligent incubation chamber to facilitate the growth of our mushrooms in a controlled environment. We hope to achieve this by using a temperature and humidity sensor with a feedback control loop.
-Rebecca

July 11th
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Minipreps were made from the crtY plates, from which a digest screen was prepared using Not-I to see if the ligation worked. The digestions were run on a gel and some appear to have worked (X/P/S, X/P/A #2/3, and X/P/S/A #⅔ worked). Also, a glycerol stock of nptII BB was made and miniprepped, but the concentration was extremely low, so an overnight culture was made from the glycerol stock to be miniprepped tomorrow. Another transformation was done using the remaining miniprep of nptII BB from earlier. Cultures were made for Gibson #3, and a colony PCR perfomed, then run on a gel. Non-GC fragments from Gibson #3 were too small, but GC #⅖ were good (1kb fragment), and #4 had a band at 1kb with another smaller fragment. Finally, nptII BB was digested with X/P then column purified, and PtrpC was digested with S/P then gel purified.

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

July 12th
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Today nptII BB and PtrpC were quantified. Also, nptII BB transformants were miniprepped, and another glycerol stock of nptII BB was made. The DNA for Gibson Assembly was miniprepped, and GC colonies 2,4, and 5 from the Gibson #3 gel yesterday were prepared for sequencing. NptII BB was digested with the PtrpC digest from yesterday. More cultures of X/P/S #1/2/3, X/P/A #⅔, and X/P/S/A #⅔ were made. CYM medium was created, and PC13Z was transformed from a kit plate.

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

July 13th
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pCg13Y was desalted, transformed, then plated into E. coli. Antibiotic stocks G418 and PPT were made (100x). G. lucidum cultures were inoculated with varying levels of antibiotic to test the strain's sensitivity for future selection purposes.

Today we worked with fungi! We applied different amounts of antibiotic to G. lucidum, to test the sensitivity of our strain of fungi. In addition, cloning of pCg13Y was nearly completed.
-Rafael

July 14th
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Colony PCRs and cultures of pCg13Y were completed to determine the success of cloning nptII BB downstream of PtrpC. Unfortunately, a gel revealed that the dna fragments appeared at 0.6kb instead of 1.4kb. pC13Z and pAK13AD were miniprepped, but had to be thrown out due to contamination of our EB. New cultures were made to be miniprepped again. A glycerol stock of PC13Z was made, too.

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

July 14th
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{{{tech}}}

This week, drylab split up so members could focus on their strengths. Rebecca and Sara began designing our feedback controller, with some much appreciated advice from the previous drylab team leader, Dan. Ritvik, Manny, and Mac began coming up with design considerations for the incubation chamber. We were most concerned with size; too small and it wouldn't be useful, too big and it would be difficult to control any variables. We eventually decided that a 1x1x2' chamber would probably be a good compromise. Rebecca, Sara, Ritvik, and Mac also attended machine shop training this week. In order to complete our training, we each had to construct a small aluminum lamp using a milling machine and a lathe. Fortunately, everyone emerged from the training session one lamp richer, and with a full set of fingers on each hand.
-Mac

Week 5

(07/15 - 07/21)

July 15th
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{{{tech}}}

Rebecca looked into the components of the temperature feedback control; namely, the temperature sensor, the microcontroller and the heating element. Put in an order for the temperature sensor, microcontroller (an Arduino Fio) and Xbee ports for wireless communications.
-Rebecca

July 15th
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Due to the contaminated EB found yesterday, our EB supplies were checked to ensure no further delays. Gibson hph was submitted for sequencing, and pAK13ADs and pC13Z(GFP) were miniprepped. PC13Z, PC13F, PCP13X, PC13M, and PC13E were PCRed with primers 31 and 32, the standard pSB1C3 primers. A gel was run to check the PCRs-all looked successful except pC13M and pC13E which had nonspecific bands around 1kb. A new technique was used in the following digests, which used a restriction cocktail of enzymes to cut the dna. PAK13AD was digested with E/S/SacI/ClaI so that it could be placed in PSB1C3 using SpeI instead of PstI due to the small distance between the PstI and the SacI cut sites. PC13F was digested with E/S/KpnI so PAK13AD can be placed in it. PAK13D was digested with S/P so crtI/crtB can be placed in it. All the digests were then column purified rather than gel purified--a move which has improved our ligation efficiencies. Then the purifications were quantified. Cultures were made for PC13P and PC13K. Finally, PAK13AD was dephosphorylated then ligated into PC13F (PC13AH).

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

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

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

July 16th
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Sequencing results came back positive for the Gibson cloning of hph, so a culture of gibson colony 5 was made. PC13AH was transformed and plated, and pC13K and pC13K were miniprepped. pCp13X (S/P), PC13F (S/P), PC13Z (S/P) were purified and digested along with PC13P (X/P) [also dephosphorylated], then all were column purified. Reran a PCR of PC13M and PC13E, and one of PC13K with primers 31/32. All PCRs were cleaned, then run on a gel along with PBARGPE1 and PBARGPE1 which had been digested with NotI today. Results: PC13E had a 3kb fragment (good), PC13K had a .9kb fragment (should be 1.7-1.8kb), PC13M had a 1.2-1.5kb fragment (good), pBARGPE1 had a 4kb fragment (super-coiled) and pBARGPE1 with NotI had a 5-6kb fragment (has a NotI site-good). From these results it can be concluded that everything worked except PC13K. Ligations of PAK13D to inserts PC13Z, pCp13X, and PC13F along with vector PC13P to inserts PC13Z, pCp13X, and PC13F were performed. Also, overnight digestions of PC13M, PC13E, and PC13K with X/P were set up. Transformation was attempted for PC13AI and PA13AJ, but the cuvettes weren't cleaned properly so it failed. Finally, our dwindling supplies prompted the ordering of more Spe1 and membrane filters.


-Rafael

July 18th
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All 18 (yes, 18!) of the pC13AH colonies that had been screened by colony PCR yesterday were miniprepped. We digested the miniprepped colonies of pC13AH clones (T7 promoter with crtY in pSB1C3) with NotI to see if the restriction cocktail method of cloning worked. On the gel that was subsequently run, bands were expected at ~2.1 for pSB1C3 and ~1.2 for crtY+pT7. The restriction cocktail method worked! The bands appeared at the correct lengths on the gel for pC13AH. There was not enough miniprepped DNA for pC13AH to send for sequencing, so the colonies were cultured again for another miniprep. None of the ligations into pAK13D (pSB1AK8 + PT7) worked -- pC13Z (GFP), pC13M (crtB), and pCp13X (bar) all failed => try cloning only using restriction cocktail method, instead of attempting PCR with VF2 & VR. Digested pC13Z, pC13M, pCp13X, pC13K (crtI) with XbaI and PstI in addition to ApaLI and SacI for later ligation with pAK13. pAK13D had been previously digested with SpeI and PstI, column purified, and dephosphorylated for insert ligation. More pAK13D was grown to acquire more DNA to be miniprepped tomorrow. Nupur performed colony PCR and made cultures of transformants from yesterday. All transformations appeared to have very low efficiency--perhaps an issue with heat shock stocks? Transformations of AI-AL (mRFP cds and lox site parts) all yielded colonies, though at really low efficiencies compared to most transformations off of kit plates. These were screened by colony PCR to verify. Ligations into pC13P all yielded colonies, but at very low efficiencies. These were pC13E (T7 pol), pCp13X (bar), and pC13Z. All were screened by colony PCR as well. Taq was used to perform 5 colony PCRs, then all colony PCRs were run on a gel. Also, pCg13s (nptIIBB)was digested with XbaI, PstI, ApaLI, and SacI for later ligation to pC13P, which had been previously digested with SpeI and PstI, column purified, and dephosphorylated. Overnight ligations of pC13Z, pC13M, pCp13X, pC13K, pCg13S with vector pAK13D were set up, along with overnight ligations of pC13K and pCg13S in vector pC13P.

The restriction cocktail method of cloning worked! Cloning of pC13AH (T7 + crtY) was shown to work using this method. We are also continuing our cloning of the crtI and crtB pathways.
-Danielle

July 21st
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{{{tech}}}

This week, the fabrication group focused on what materials to construct our chamber from. At first we considered a single wall design, but this was eventually scrapped, as we were unable to find any material that would provide sufficient insulation at a reasonable thickness (and cost). After putting our heads together, we decided on a double wall with an insulating layer in between them. Mac began drawing up the chamber in Solidworks.
-Mac

Week 6

(07/22 - 07/28)

July 23rd
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{{{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 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 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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{{{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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{{{tech}}}

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

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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{{{tech}}}

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

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 10th
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{{{tech}}}

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

Week 9

(08/12 - 08/18)

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 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 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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{{{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

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, ran 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 G. lucidum 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 Cochliobolus 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 G. lucidum transformation in the future. We will also check for homology between the promoter gpd in G. lucidum and Cochliobolus to make sure we are using DNA that can be expressed. Our new strategy will be to insert genes after Cochliobolus 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 G. lucidum behind for now and work with Cochliobolus, 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 G. lucidum 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 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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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 13th
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{{{tech}}}

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

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

Week 14

(09/16 - 09/22)

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 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

Week 15

(09/23 - 09/29)

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 E. coli. A culture of G. lucidum was diluted and homogenized, and 150 μL aliquots were spread on plates and let dry. Paper discs were soaked in 100X solutions of Geniticin, Hygromycin, Ampicillin, Kanamycin, and Chloramphenicol and placed in the center of each plate. We miniprepped holin 8 & 9. The results from the growth assay do not look good. However, we found that the T7 polymerase in BL21 is controlled with an IPTG inducible promoter, so more pC13BA was grown with IPTG and plates were all re-inoculated with the corresponding amounts. An appropriate volume of LB was also added to some plates to account for mass changes. The zone of inhibition tests were also restarted with 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 E. coli for characterization, and set up a zone of inhibition experiment to test the efficacy of antibiotics and antifungals on G. lucidum.
-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 needs to 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 E. coli 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 Aspergillus 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 E. coli. However, there was normal growth around the 10X hygromycin discs for the BL21 E. coli 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 E. coli.
-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