Team:UIUC-Illinois/Notebook/MeetingNotes

From 2012.igem.org

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7/16/12 Monday
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Reflection:
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Optimistic goals:
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*By the end of the summer, 4CLSTS and BM3 and mutants BM3 tethered with PUF for the scaffold project and biobrick TAL
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*Entrepreneurship: Concern about the project progress. Send out our constructs for Patenting.
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*Necessary PUF synthesis if PUF doesn’t get cloned by the end of July. Thinking about mathematics modeling. 
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*We will win the Best Hail Mary Award!
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Tuesday, 7/17/12
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- Uros - By tonight will have a google doc up of his plan for the primers for the tethering and making the split CFP construct (in parallel). There was a mistake in one of the primers, but he caught it. Received an email back about split GFP, and got referred to another person. Will email her tonight and hopefully will get a sequence. If cloning/tethering doesn’t work by Monday, will send out constructs for sythesis.
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- Anthony - still working on putting WT PUF in pBAD. Ran a gel and extracted from yesterday’s digests. There’s no band for pBAD. So starting 3 new digestions from different minipreps of pBAD. Hopefully will get some result from that, because can’t start ligation until the pBAd digest works.
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- Asha - old plates of YFP constructs had weird growth 4 days. Uros and I looked at them and didnt think that they were fluorescing. Working on a positive and negative control to look at fluorescence. Negative will DH5a and positive will be E0030 subcloned into ptet (working with Angela on that.)
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- Divya - working on biobricking WT PUF. PCRed more WT PUF. Isiah will run the gel on them tonight. Also poured Amp and CM plates (10 of each). Still waiting for the alkanes to arrive, so the Washington characterization is delayed. Isiah will call the company to see what is taking so long.
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Thursday, 7/19/12
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- Uros - no meeting yesterday because Uros and Angela discussed the format of the daily meeting. A big reason why the meetings run so late is because people are late. JUST BE PUNCTUAL! If you have a legit reason for being late, text everyone to let them know. If you miss a meeting look at this google doc to look at everyone else’s work and also be sure to update the google doc for the lab notebook. Come in tomorrow with a brief checklist of your project goals for the end of the summer so that everyone can be updated and be ready to start participating in quality discussions about all the separate projects. The google doc on which to share these summaries has been shared with everyone.
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- Uros - trying to make a split CFP construct, trying to clone the 2 parts (C and N terminus) into the plasmid. The Cterminus part worked today for PCR. Need help with this part tomorrow. Adi and whoever else needs to be in lab tomorrow at 9 to help out. Uros will send out a thorough google doc of instructions. Made a final draft for the WT PUF alpha g for sythesis. Upon advice of a post doc made a draft for WT PUF and the split GFP tethering.
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- Divya - PCR of WT PUF yesterday. Isiah ran the gel. That PCR procedure worked. Another PCR today was performed by Isiah according to a diff procedure. The gel was run by Divya and the PCR failed.
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- Anthony - transformed and plated yesterday’s ligation, started another ligation in case those don’t work.
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- Angela - negative control for YFP is cloned, YFP construct is completed, sequenced and it fluoresced. Successfully used primer extension to clone Venus YFP into protet. Instead of using the biobrick E0030, we will use Venus from now on. Now we will be able to start looking at our designed RBS to see if it works. Will get primers on Tuesday or Wednesday. Takes 2 days to clone via primer extension.
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- Bob - been helping several people out on all of the projects, but his main efforts are for the PHAT project. Wiki design question: for the lab notebook, we are split into the PUF notebook and the PHAT notebook. the PHAT section is structured in a continuous page where the tab buttons jump you to a different location on the page. The PUF section is structured so that every tab has a different page. Which format is preferred?
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- Cara - PCR that Bob performed yesterday was run on a gel. Think it worked, but the ladder wasn’t really clear, so the digestions are being run with lots of controls. Will also soon be able to see if the BM3 site was accurately mutated to get rid of the PstI site. Do we need primers to send it out for sequencing? . Has the 2 primers for the outside of the 4CL:STS insert, but needs primers for the inside of it. Perhaps a colony pcr would be best. Use VF and VR2 standard primers. However, need to transform and plate to get the colonies necessary for that (have been working from liquid cultures so far.) Will also make IPTG.
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- Adi, Entrepreneurship - talked to Joe and Alex about continued wiki design. First image will be the photo of our team, then it will slide by to be a video of the elevator pitch. Doesn’t want it to be a video of people, but more of an animation or a pretzi-like thing. Will have voice try-outs in collab with human practices video. Is compiling a new list of supplies.
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- Asha - gonna move on to cloning wtPUF to PBAD30 with Anthony. Human practices is going to do filming soon. Need microphone. Anthony has a professional recorder.
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Friday, 7/20/12
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- no advisor’s meeting
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- Meetings have been moved - they will only be on Monday, Wednesday, and Friday. On Fridays that we have advisor’s meeting, we will not have a separate meeting but instead wrap up for a few minutes after the advisor’s meeting.
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- For future gels, or really nice gels that we want to take good picture of, don’t use the big UV tray next to the computer.
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- Angela - YFP (Venus) fluorescence! Also, it’s sequence has been confirmed.
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- Divya - did PCR and will run gel later.
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- Asha - PCRed more WT PUF for pBAD.
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- Isiah - working on digestion of linear PSB1C3. Will later run a gel.
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- Adi - miniprepped petduet and the K---- biobrick part (NCFP and CCFP).
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- Cara - Waiting on the digest for more conclusive results
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- Uros - tethering is delayed because a delay on one primer and no sequence of mutant PUF.
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Wednesday, 7/25/12
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- Angela - no colonies on PUF eYFP construct. Needs minipreps of puf, pbad, ptet, everything. Did a 4th ligation and will hopefully see colonies. the backup is synthesis, will speak with Dr. Jin about it. Credits! We can get credits for this research, but need to check with the individual advisors in your home department. An evaluation has to be done after the summer, but should really been done in the first 2 weeks of the semester. Will probably get 2-4 hours. Need to get going on biobricking and characterizing!
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- Asha, human practices - will hold interviews with Dr. Mumm and Dr. Below for the transgenic plants video. Will also refine the media research from the beginning of the summer to create a database. Still trying to trouble shoot pBAD problems. Will digest minipreps from inoculation tomorrow and will use butanol precipitation on ligation on Friday morning.
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- Divya - met with Melissa and the IGB communications director Nick Vassi yesterday. Discussed creating a video and writing an article. Nick Vassi said that if we were to write an article, he would review it and send it to his contacts at the Daily Illini. For the video presentation, he said gather as much footage as possible. Will present this video for recruitment among bioe 120, eng 100, and las 199 classes. For high school presentations will use the human practices videos. Starting to think about contacting these high schools and classes. Change in the plan, Divya will focus on the Washington characterization, Isiah will do the PUF biobricking.
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- Anthony - transformations from heat shock didn’t work. Desalted and electroporated again and hopefully will see colonies tomorrow. Started another ligation just in case that doesn’t work. Also, everyone should be sure to put away all of their supplies.
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- Ligation troubles - is it reagents or the materials?
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- Bob - Continued a restart of the BM3 fermentations, now with a control that is not induced. Did colony PCR of 4CL:STS. Want to discuss it with Angela because don’t know how to interpret the results.
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- Cara - tried to make comp cells, but need an id and access code to run the big centrifuge and need to talk to brad about that. Will look into it and then make the comp cells as soon as possible. Last piceatannol synthesis, did something wrong. Might have overloaded the TLC plate and also had a weird substance left over from the speed vac. Will also try to further refine the protocol. Next step is to lyse the cells to see if the cells are creating picetannol, and just not exporting it. After that will need to try to purify the supernate to try to get some concentrated supernantent to run.
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- Uros - trying to tether PUF and CCFP. Yesterday tried to amplify the 2 parts but was only using forward and reverse primers (1 primer for each). This amplified PUF and kinda worked on CCFP. But it didn’t work for the actual tether. So tried again today just with the tether with HF buffer instead of GC buffer. Got primer dimers again. Ran it again on a gradient of 50-55-60-65, used less primers, lengthened annealing time. Will check the results of that later tonight. If this fails the next plan is to do PCR so that the PUF and the CCFP will have the restriction site and linker sequence on them even before tethering. Will be gone July 30-Aug. 6 and needs someone to help out with the RNA scaffold characterization. Will send out a google doc to everyone by next week. Waiting on Angela to get the mutant PUF sequence to get sent out for synthesis in case this doesn’t work.
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- social! Thursday night there is a rodeo at 7 at the county fair! It’s $5 to get in the door and Cara will post more info on the fb page. consider eating before because the food is expensive.
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Monday, 7/30/12
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- Adi - needs to figure out how to pitch to companies with whatever data we have so far. Cara and Angela should send Adi a three sentence summary of preliminary work and data to convince companies to sponsor us. Do this by Thursday.
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- Bob - been working on the petDUET and CCFP stuff for Uros. The ligation was finished over the weekend, so transformed into DH5a today. Also the petDUET miniprep DNA has been yielding really low, so inoculated 16 mL to get a good supply.
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- Cara - will be focusing on her final on Wednesday. Will resume work on Thursday. Might be considering sequencing.
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- Asha - got colonies from last pBAD-wtPUF ligation, think it’s the nuclease free water. Gave to Angela for colony PCR. Made inoculation for glycerol stock of new, better pBAD. Had interview with Dr. Below, got 30 min of footage.
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- Anthony - started new digestions for pBAD-wtPUF.
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- Angela - no meeting on Friday. Used the new nuclease free water (which appears to be working well!) Have to order new primers for the PUF-binding YFP construct. Will be gone Wednesday, Thursday, and Friday. Barrier to ordering Uros’s tethers - doesn’t have the mutant sequence from Dr. Wong.
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- Divya - replated transformations of WT PUF biobrick, Isiah did a colony PCR that might have been successful but will redo it tomorrow. If it is confirmed, the PUF biobricking is complete. Will be continuing on with the Washington characterization tomorrow, talked to Jin’s lab about GCMS.
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Monday, 8/6/12
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- Angela, PUF - sequencing results are not back yet. Perhaps the concentration was too low. Cotransformations have been performed and obtained colonies. The first protein test was inconclusive, but the colonies don’t fluoresce.... perhaps the YFP has become degraded. Ended up characterizing E0030 by mistake, because out of nowhere it works. Will be working on lac Z characterization as well.
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- Anthony, PUF - did a protein gel to analyze WT PUF expression. Under arabinose, it looks that there is not much expression. Transformed into BL21 to see if that works better. Will test DH5a once more, and if that yields the same result than the BL21 will be used. The problem could be the T7 promoter.
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- Arabinose problems - Angela researched LB and it’s very rich, probably has arabinose. So will need to use M9 instead because it won’t have arabinose.
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- Asha - completed all the interviews for human practices, will work with Divya to tie human practice to publicity. Will go home and edit videos like crazy =)
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- Divya - working on an article for the daily illini. Will be trying to coordinate a practice session that will also function as a seminar for critique. Will invite IGB, general public, and BioE department. Also, quad day!
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- Divya, characterization - ran a control of the GCMS today. Need to confirm that the controls worked. needs someone else to run the rest of the tests on the GCMS tomorrow. The tubes are all prepared and it’s really easy. It will take about an hour. Adi will take care of it.
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- Adi - is now updated with the PUF project, and now needs a PHAT project update. Needs them to work on the business plan.
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- Bob - has been helping out with Uros’s stuff. Needs to redo colony pcr. But doesn’t have a lot of colonies left on the original plate, so inoculated from the leftover colony and will replate and redo colony pcr.
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- Cara, PHAT - this week will finish PCR’s for TAL, is ready to send out 4CL:STS for sequencing (1.8 KB gene, has 5 sets of primers), has performed mutagenesis on BM3. Emphasis will be on thoroughly characterizing the BM3.
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Friday 8/10/12
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Angela:
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- Successfully transformed two plasmids in the cell and this has been confirmed
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- Have tested the expression of PUF in the cell
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- The YFP that has been cloned functions as expected
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- The artificial RBS does not work as well as the native RBS
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- Has been in contact with Dr. Wong, will be on contact with him on Sun about mutant PUF
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Anthony:
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- Will be testing the fluorescence of all the combinations of the controls and testers
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- Hoping to have the combinations tested by Sun/Mon.
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Bob:
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- Has been changing things on the wikio to reproduce what Calgary had on their wiki
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- Change wiki background
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- Working on making sure the changes to the wiki display the same in each browser
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- Working on making an interface that is clickable and will link to other parts of the wiki from the main page based on each project of the team.
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Cara:
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- Did PCR of TAL, will use PCR clean up then run on gel to determine size of the PCR fragment generated
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- Performing overlap extension PCR to splice together pieces of BM3 gene that have the PstI site removed
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- Making TB media to grow BM3 for characterization assays
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Uros:
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- Sent Brad a draft of PUFwt tethered to alphaGFP and he responded and said everything looked good and he changed the his tag
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- Heard back from the representative for the first synthesis quote
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- Emailed Courtney regarding the quote for the synthesis and the sequences to be ordered
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- Waiting for two primers for RNA scaffold cloning to come in
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Friday, 8/24/12
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Asha - went to a conference on technical presentations, is creating the human practices video and powerpoints for outreach school visits
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Isiah - helped out with scaffold, ran a PCR that didn’t work very well, will throw out and make more media
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Anthony - been helping Angela with PUF constructs and running protein expression gels. Mutant PUF is working well and WT  PUF not so well.
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Bob - been working on restructuring the wiki alot, needs all the new/revised protocols,
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Uros - someone needs to throw out the contaminated stack of Amp plates in the 4 degree room and make new ones, been doing scaffold work, got successful transformations of the scaffold, but had problems with minipreps (DO NOT USE the p3 neutralization buffer), used a lot of micrograms but got a big smear, will try in vitro transcription tonight, if that works will move on to running gels
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Angela - have been testing with YFP but still need more results, cloning 8 more constructs so needs a lot of help (been recloning things alot as they don’t work), will send results directly to Bob to be posted on the wiki, cloning works because florescence is observed but still need to further quantify the functionality of PUF, still need help with the YFP testing and with cloning all the constructs
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Sept 3 deadlines - need to have the final draft of the abstract and the safety questions. For the abstract need a draft by Wed/Thurs to have at least one advisor look at it. For the safety questions need “institutional” approval of our safe practices.
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Quad day - Divya and Asha working on a new poster and handouts, maybe wearing labcoats? (good or bad idea... debate)
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1-2: Isiah, Angela
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2-3: Anthony, Asha
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3-4: Bob and Cara
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Jamboree prep - Clear your schedule for Sept 26 at noon because it’s the seminar presentation at IGB.
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Friday, 8/31/12
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Should we all come together on one project or continue in these multiple tracks?
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- option 1: everyone comes together at the end to try and invest max time in PUF in order to produce results. Hopefully this not only yields results but will help team unity and bonding
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- option 2: everyone continues working on the side projects. perhaps all of them don’t get done, but there is also the opportunity to have more biobricks and more data. Everyone still had leadership and ownership over their own projects.
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- what will the judges like the most? typically they like 1 really complete and detailed project, and then a small side project or an idea that accompanies the other one.
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- PHAT: trying to biobrick 2 of the enzymes. After biobricking them, will work on characterizing them.
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- PUF: tested WT PUF with YFP. Whenever there is PUF present, YFP doesn’t glow. This answer has been affirmed 5 times. However, the control is also not glowing, but it shoudl be. Also did protein expression gels for WT PUF and mutant PUF. Found that mutant PUF has good expression, but WT PUF has little expression. Today did a mutant PUF test and found that the mutant PUF when applied to the PUF binding site YFP, did not bind. This shows the specificity of binding of the PUF molecule. Dr. Wong has also sent a help construct in order to reproduce his experiment. The construct consists of an engineered mutant PUF. It was inserted into BL21. The mutant PUF site specifically binds to LacZ. Our next steps are to transform the mutant PUF and he Lac Z testing construct to BL21. Biobricking: WT PUF and mutant PUF with plans to characterize both of these. Currently testing:
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- control YFP/wt puf YFP (protet)  with WT PUF (pBAD)
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- control YFP/wt puf YFP(protet) with mutant PUF (pBAD)
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-***control YFP/wt puf YFP (pBAD) with WT PUF (protet)
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-***control YFP/ wt puf YFP (pBAD) with mutant PUF (protet)
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-***control YFP/wt puf YFP (pBAD) with WT PUF (pet)
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-***control YFP/ wt puf YFP (pBAD) with mutant PUF (pet)
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- Lac Z (pBAD) with mutant PUF (pet)
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- Lac Z (pBAD) with WT PUF (pet)
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- RNA scaffold: characterizing the scaffold and wants to biobrick the scaffold as well. Will be characterizing the synthesized parts and also wants to biobrick the split GFP parts. That means 3 biobricks: scaffold, 2 split GFP. Yesterday got purified RNA of the scaffold (but didn’t denature, so a little uncertain). Will move onto doing gel shift assays once the constructs are obtained. Aug. 14th, send first part, Aug 17th sent second part to courtney. She canceled order because thought the 2 were the same. Ordered the parts finally on the 24th. Suggested is 10-12 business days, but will probably obtain it in mid-september. Can’t biobrick the 2 split GFP parts until the synthesized constructs are received. Uros will biobrick the scaffold on his own, but will need help when the split GFP parts come in.
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Roles to fill:
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- fluorescence testing: Angela
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- biobricking everything: Isiah, with help from Adi
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- RNA/protein gels: Uros
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- cloning (not biobricks):
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Advisors meeting
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present: Kori, Brad, Todd, Ahmet
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Upcoming deadlines: abstracts, safety questions, track selection due September 7th
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- ongoing deadlines: poster, presentation, wiki (WIKI FREEZE is Oct. 3)
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- regional jamboree: Oct. 12- 14
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Currently have: control YFP, wt puf binding site YFP, WT PUF.
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- co-transformed control YFP + WT PUF, and then wt puf binding site YFP + WT PUF in DH5a
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- results of fluorescence testing: control YFP does not glow but it should, binding site YFP does not glow either, but need to clarify the behavior of the control. (blast test reveals there should be no other PUF binding site in the YFP, so maybe the control binding site is actually not working. there could also be interference activity between the YFP and PUF).
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- have we perhaps mixed up what we think is control and puf binding site YFP?
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*** will try the PUF with mcherry instead, to see if it’s just an issue with the YFP. Also, use new cuvettes for the transformations. 
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Will try in the future:
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- subclone YFP into pBAD (was previously in protet) 
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- back up plan from UNC’s Dr. Wong: mutant PUF binding to Lac Z in BL21
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RNA Scaffold update:  
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- tried to use primer extension for a week to tether split GPF and PUF, but it failed so ordered the parts for synthesis. The parts were ordered on the 24th of August. The quote was that they would come in 10-12 business days, but it’s pragmatic to expect it will take longer.
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- Transformed RNA scaffold, prepped, and got results for in vitro transcription. Got low concentrations and a weird value from the plate reader. Did another run of in vitro transcription with better results. optimistic about new results and got pure RNA scaffold (but didn’t denature ladder and didn’t use a certain buffer for the ladder). Therefore the ladder is incorrect. However, feels confident in result because obtained a single, defined band of notable concentration. Ready to do gel shift assays when the split GFP parts that were ordered finally arrived.
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- Needs funding to be able to continue this research in the Hergenrother lab. Need acrylamide casting equipment, RNAse zap. For the future would also need to buy the nickel and tine reagents.
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- don’t buy the gel box, just need the reagents. look for a gel box somewhere around the IGB. Could also just buy pre-made gels.
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- concentration of RNA? paper suggests 1000 fold, has used 100 fold. apparently that alone is enough to see a shift. However, that can be optimized.
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- the potential to do flow cytometry. There are locations for this on campus, but they might not be bacteria friendly and an hourly fee is also required to use the equipment.
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- in the next week will try to purify mutant and WT PUF, and then do gel shift assays with them.
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- need a distribution of work for all of this.
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Writing the abstract (due Sept. 7)
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- a paragraph, similar to what would be in a paper
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- can write about things we haven’t performed yet, but need to be realistic
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Saturday, 9/15/12
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- EC announcements - funding for fall due Oct. 1, Angela needs to go to a website meeting (at the meeting will give instructions about EC websites and funding application)
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- t-shirt design from Divya - royal blue vneck with Illinois iGEM logo in upper left shoulder, sponsors on back (need to fix logo for the IGB)
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- tshirt design approved by all!
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- powerpoint - next Friday will have a practice presentation for the advisors
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- video - Bob has been working on animating through powerpoint.
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- poster design - need to graphically represent all the data and constructs (takes a lot of time!), Cara has experience with this
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Revision as of 19:19, 2 October 2012

Header

Protocols

Meeting Notes

Pre-April

  • 2/17/12
  • 2/19/12
  • 2/26/12
  • 3/4/12
  • 3/6/12
  • 3/7/12
  • 3/11/12
  • 3/25/12
  • 3/27/12
  • April

  • 4/1/12
  • 4/3/12
  • 4/4/12
  • 4/8/12
  • 4/14/12
  • 4/15/12
  • 4/17/12
  • 4/22/12
  • 4/24/12
  • May-July

  • 5/1/12
  • 5/25/12
  • 5/29/12
  • 6/1/12
  • 6/11/12
  • 6/12/12
  • 6/18/12
  • 6/19/12
  • 6/20/12
  • 6/21/12
  • July-August

  • 7/5/12
  • 7/6/12
  • 7/9/12
  • 7/10/12
  • 7/11/12
  • 7/12/12
  • 7/16/12
  • 7/17/12
  • 7/19/12
  • 7/20/12
  • 7/25/12
  • 7/30/12
  • August-Jamboree

  • 8/6/12
  • 8/10/12
  • 8/24/12
  • 8/25/12
  • 8/31/12
  • 9/15/12
  • Select a date to read respective meeting notes.


    Retrieved from "https://2012.igem.org/Team:UIUC-Illinois/Notebook/MeetingNotes"