Team:UIUC-Illinois/Notebook/MeetingNotes
From 2012.igem.org
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- | <center><h2>April | + | <center><h2>April</h2></center> |
<li><a name="meet10" >4/1/12</a></li> | <li><a name="meet10" >4/1/12</a></li> | ||
<li><a name="meet11" >4/3/12</a></li> | <li><a name="meet11" >4/3/12</a></li> | ||
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<div id="meetingselection3" class="meetingselection"> | <div id="meetingselection3" class="meetingselection"> | ||
- | <center><h2> | + | <center><h2>May-July</h2></center> |
- | <li><a name="meet17"> | + | <li><a name="meet17">5/25/12</a></li> |
- | <li><a name="meet18" > | + | <li><a name="meet18" >5/29/12</a></li> |
- | <li><a name="meet19" > | + | <li><a name="meet19" >6/1/12</a></li> |
<li><a name="meet20" >TBD</a></li> | <li><a name="meet20" >TBD</a></li> | ||
<li><a name="meet21" >TBD</a></li> | <li><a name="meet21" >TBD</a></li> | ||
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</div> | </div> | ||
<div id="meet19" style="display:none"> | <div id="meet19" style="display:none"> | ||
+ | <textarea rows="30" wrap="soft"> | ||
+ | iGEM Advisor’s Meeting | ||
+ | 5/25/12 | ||
+ | PPT Presentation | ||
+ | Biobrick with PUF binding site | ||
+ | - Grow on Xgal to test blue/white colonies rather than quantifying LacZ | ||
+ | - Need a plasmid without Lac I already. Easiest way is to get the strain from the | ||
+ | people who made that biobrick already. A different option is to do a Lac I | ||
+ | knockout. | ||
+ | - Rao: Faster and easier to just do GFP expression. | ||
+ | o A blue/white screen is just a yes or no. It’s not a quantitative assay. | ||
+ | o Can do LacZ and GFP in parallel. That’s a stronger experiment. | ||
+ | Potential Plan | ||
+ | - Assemble biobrick | ||
+ | - Then introduce PUF binding site and mutated PUF binding site. Insert before | ||
+ | RBS of Lac I, after RBS of Lac I, | ||
+ | - Assemble last biobrick of PUF+PIN | ||
+ | - The biobrick that we intend to make is made of smaller biobricks that we will | ||
+ | hopefully process all in parallel. | ||
+ | - Different assembly methods? | ||
+ | - Rao: forget fancy PCR things, including Gibson assembly. | ||
+ | o Need a simple control to test the PUF. GFP is a 2 week control | ||
+ | experiment to test PUF levels. | ||
+ | o The modular Lac I/Z system is worth pursuing, but it should be done in | ||
+ | parallel with the GFP experiment. | ||
+ | - The terminators are small, they are going to be hard to cut out of a gel. How | ||
+ | will we tackle small segment assembly? | ||
+ | o 3A assembly is made so that we don’t have to do gel purification. | ||
+ | Therefore we will be fine on the promoters, RBS, and terminators. The | ||
+ | hard part is the PUF sites | ||
+ | o A possibility is to biobrick PUF by itself so that we can simply order | ||
+ | primers and such for it. | ||
+ | o Other option: ignore the existing biobrick BBa_Q04121 and simply put | ||
+ | the RBS and PUF binding site on the forward primer for the reporter so | ||
+ | it’s all together to begin with. The reverse primer would have both the | ||
+ | terminators. | ||
+ | Constraint – RBS must be no more than 10 base pairs away | ||
+ | from the start ATG. The PUF binding site will be better suited to | ||
+ | before the RBS | ||
+ | Also, we can expand the biobrick with the addition of each | ||
+ | primer, by adding aroud 40 bp everytime | ||
+ | Biobricks | ||
+ | - PUF is too small to add on its own. Would need to put PUF with the RBS or | ||
+ | something. | ||
+ | - Ideal biobrick: RBS, PUF, and Lac I reporter all together | ||
+ | - It’s too small to biobrick the PUF binding site | ||
+ | o Really makes a problem with creating a PUF toolkit | ||
+ | Final Plan | ||
+ | - PUF binding site with Lac Z | ||
+ | - Mutated PUF binding site with Lac Z | ||
+ | - PUF binding site with Lac I | ||
+ | - Mutated PUF binding site with Lac I | ||
+ | - PUF binding site with YFP | ||
+ | - Mutated PUF binding site with YFP | ||
+ | - PUF+PIN | ||
+ | - Mutated PUF + PIN | ||
+ | - The RBS and PUF binding sites will be in the promoter for the varying gene. | ||
+ | That simplifies putting that tiny sequence into E. Coli | ||
+ | Remaining questions | ||
+ | - How extensively should things be characterized? | ||
+ | o Worry about it when we get there. | ||
+ | - Should we link PUF to another protein domain on top of the endonuclease? | ||
+ | o Don’t worry about it, we need to basics first. | ||
+ | o We have 3 strains of PUF: Wild type PUF (just PUF), PUF with | ||
+ | endonuclease, PUF with mutated endonuclease | ||
+ | PHAT project | ||
+ | - Korean lab is making stocks of stuff and preparing to ship them early next | ||
+ | week. Will hopefully get them on Wednesday. We will also receive a plasmid | ||
+ | map. | ||
+ | o Need to get them a FedEx account number because normal mail will | ||
+ | take weeks | ||
+ | - First step is to biobrick the wild type BM3 (cytochrome P50), and 2 other | ||
+ | mutants (one with the highest activity level and the other with the highest | ||
+ | longevity) | ||
+ | - Second step, redo their assay to make sure that biobrick formatting doesn’t | ||
+ | affect the enzyme’s activity | ||
+ | o How do we measure piceatannol? We don’t have that capability to run | ||
+ | the mass spec. | ||
+ | o Look into measuring via engineering or some way other than | ||
+ | machinery. Look at reduction reactions. Send an email to the Korean | ||
+ | professor to see if they used any other methods. | ||
+ | - Next step, order powder resveratrol. Mix it in the media and see if piceatannol | ||
+ | would form. | ||
+ | o Contact Matthew Koffas because he published about producing | ||
+ | piceatannol in E. Coli. Tell him that despite patenting issues, we just | ||
+ | want any strain for a proof of concept idea. | ||
+ | - Still looking to play around with the chemical drawing software. It would be a | ||
+ | nice theoretical presentation at iGEM. | ||
+ | Announcements | ||
+ | - Isiah: Will start using a virtual lab notebook that everyone should write when | ||
+ | they use something up. There will be a piece of paper on the bench, but | ||
+ | digital is better. | ||
+ | - Daily Team meeting at 5 in the Union basement. | ||
+ | - Advisor’s meeting every Friday from 3-4 PM. | ||
+ | </textarea> | ||
</div> | </div> | ||
<div id="meet20" style="display:none"> | <div id="meet20" style="display:none"> | ||
- | + | <textarea rows="30" wrap="soft"> | |
+ | iGEM daily meetings | ||
+ | Week of 5/29-6/1 | ||
+ | 5/29 | ||
+ | - No work today because of MMG accident. | ||
+ | - PHAT project: switching to a tyrosine pathway that is cheaper and more | ||
+ | suitable for e. coli. Mark (Octochem) is buying us some resveratrol. If | ||
+ | pursuing a bottom up building model, biobrick resveratrol separately because | ||
+ | there is a distinct demand for it. | ||
+ | o Timeline: End of June, have 2 biobricks of the stuff from Koffas’ lab | ||
+ | (4CL. STS, 4 kumarate ligase) (PUCO is the vector where they are | ||
+ | both already ligated together.) going pkumarate -> resveratrol -> | ||
+ | picetannol | ||
+ | o In vivo assays to confirm Korea’s results and then move on. But are | ||
+ | currently needing to cut down from 9 constructs. | ||
+ | - Melissa has contacted Richard Powers on behalf of iGEM | ||
+ | o He’s at Beckman we should go talk to him | ||
+ | - $ 95 pass to get film stuff from art department for the whole semester. Art 250 | ||
+ | and we need a separate mike. | ||
+ | - 2010 uiuc igem team has a good synbio video. Take a look at it. | ||
+ | - Entrepreneurship: still don’t know if one or 2 projects. Come up with a | ||
+ | marketing pitch, a team description, and then an elevator pitch. | ||
+ | - Publicity: Divya will make a brochure by the end of the week | ||
+ | - Journal Club: We will have one! It will probably be 30 minutes a week on a | ||
+ | biweekly basis. It is strictly optional, but there are definite benefits for | ||
+ | attending. The first one will be hosted by Angela. | ||
+ | 5/30 | ||
+ | - Lab Manager: Our inventory is now on a google doc. Whenever you | ||
+ | completely use something, make a note of it! | ||
+ | - Brochure is in progress. | ||
+ | - Entrepreneurship: Adi is going to start contacting companies about | ||
+ | sponsorships. Is going ahead to go and make 2 projects. Adi will also start the | ||
+ | business plan and elevator pitch with advisor Joe Bradley. He will also skype | ||
+ | with Alex about the website at some point. | ||
+ | - Webmaster: Bob talked to Bhalerao. The server is off and in his office. He will | ||
+ | make accounts for access. | ||
+ | - Publicity: Courtney has sent out a template from last year’s brochure. Divya | ||
+ | will update it for us by the end of the week. | ||
+ | - Angela: Will send us abstracts for editing and critique. This will go in the | ||
+ | brochure. | ||
+ | - PHAT: need to get the actual MTA from Koffas. Rao also wants to talk to | ||
+ | Koffas about that. Prof. Ho will be shipping the constructs on Friday, and we | ||
+ | can anticipate their arrival on Monday. Brad suggests doing TCL (thin layer | ||
+ | chromatography). Quantitatively, it will show a big or littler amount, but not | ||
+ | much else. It takes around 45 minutes. We can look into collaborating with | ||
+ | another lab to purify picetannol if necessary. We can also do LCMS or GCMS. | ||
+ | - Angela: Design the forward and reverse primers for the PUF constructs with | ||
+ | Lac I, YFP, and Lac Z. | ||
+ | - Asha: made LB, made electrocompetent cells, did inoculations for PUF | ||
+ | - Divya: made LB, made electrocompetent cells, started brochure | ||
+ | - Uros: transformed PSB1C3 | ||
+ | - Adi: met with Courtney, met with Joe Bradley, inoculated for PUF | ||
+ | - We will work in DCL on the weekends. We need to do PCR so we will email | ||
+ | advisor Ting Lu about getting space in his lab. | ||
+ | 5/31 | ||
+ | - Divya: working on brochure and blog. iGEM teams are following us and we | ||
+ | are following them on twitter! Redesigning a logo | ||
+ | - Can start thinking about a t-shirt design | ||
+ | - Bob: put a drop down menu on the wiki, changed the project page, can put | ||
+ | the twitter feed on the website under outreach | ||
+ | - Cara: emailed out new primers and would appreciate everyone’s feedback | ||
+ | - PHAT project: got sequences of P10 and wild type | ||
+ | - Angela: can use other cloning methods, 3A assembly is outdated and primer | ||
+ | extension has been recommended, for the testing of PUF binding can use the | ||
+ | native plasmid backbone and not just PSB…, it is recommended that we send | ||
+ | things in for DNA synethesis – iGEM is about the design and not the labor of | ||
+ | cloning again and again (Dr. Jin says that if we can prove it works and cloning | ||
+ | is under $500 then we should send it in for synthesis), all of these backup | ||
+ | plans are very beneficial, new PUF primers are coming in soon (estimated | ||
+ | Monday), we need an application for our project! | ||
+ | o Possible application: PUF can be used for identifying and pinpointing a | ||
+ | gene, we could try to combine the PHAT and PUF projects by using an | ||
+ | mRNA sequence with multiple PUF binding sites. The different PUFs | ||
+ | would be linked to different enzymes creating a biological conveyor | ||
+ | belt for faster picetannol production. And then you can control when | ||
+ | this mRNA is being made. | ||
+ | </textarea> | ||
</div> | </div> | ||
<div id="meet21" style="display:none"> | <div id="meet21" style="display:none"> | ||
- | + | <textarea rows="30" wrap="soft"> | |
+ | iGEM advisor’s meeting | ||
+ | 6/1/12 | ||
+ | PUF | ||
+ | - The last powerpoint for the plan was using the biobrick format, backbone, | ||
+ | terminator etc. | ||
+ | - We can use a different plasmid for testing though. So we will clone the | ||
+ | expression construct (PUF) that is already in the PET 4.3 structure into the | ||
+ | BL21 strain. Our testing plasmid will have the RBS-PUF binding site-YFP. | ||
+ | This will be a quicker test because the plasmid already has a promoter and | ||
+ | terminators. The primers for these are already order and will arrive on | ||
+ | Monday. The primer includes the RBS, PUF binding site, and restriction sites. | ||
+ | We are cutting with EcoRI and SpeI. | ||
+ | o PET 4.3 is Amp resistant, the protet is Cm resistant. | ||
+ | o We have to check the compatibility of the plasmids. Check this by | ||
+ | looking at the ORI. If they have the same mechanism that will be an | ||
+ | issue (competition and one will win over the other). | ||
+ | o There are biobricks to make this as well, but our overall goal is for | ||
+ | testing to be as quick and easy as possible. | ||
+ | PHAT | ||
+ | - Working on a gene operon to convert tyrosine all the way to picetannol. | ||
+ | - We’ve found that the 2008 Rice team did tyrosine to resveratrol in yeast. But | ||
+ | not all of their biobricks are characterized, so they may not be functional. But | ||
+ | the 3 enzymes are in the registry. | ||
+ | o Try to get these from the registry and try to put it in e. coli. These | ||
+ | probably won’t be in the kit plate, so will order from the registry. | ||
+ | o PCR the genes and build the operon ourselves. That saves the hassle | ||
+ | of the MTA because Koffas might not approve putting his stuff in a | ||
+ | biobrick. | ||
+ | o Rao will still follow up on the MTA but it might not go through. Putting | ||
+ | things into a public registry kind of defeats the purpose of the MTA in | ||
+ | the first place. | ||
+ | - The correct sequences have been obtained from Dr. Kim. All 3 BM3 genes | ||
+ | are 3 KB (big!). So are 4 genes all going to fit in one plasmid? What options | ||
+ | do we have to deal with this? How big is too big? | ||
+ | o For a standard plasmid 10,000 is the max. After that you have to go to | ||
+ | special plasmids (bac). The thing about bac is that they’re single copy. | ||
+ | o Could we take the ORI from bac and put it in a PSB backbone? No, it’s | ||
+ | not that simple. There are other factors. | ||
+ | - If we assembly the operon, it will still be novel. | ||
+ | - NEW IDEA – BIOLOGICAL ASSEMBLY LINE: we have an mRNA that is | ||
+ | never meant to be translated, it is merely a scaffold for tons of PUFs. It has | ||
+ | various PUF sequences that are all linked to different enzymes that are part | ||
+ | of the tyrosine picetannol pathway. That way all of the enzymes end up | ||
+ | close together, increasing the efficiency and speed of picetannol production. | ||
+ | o Discussion with Bhalerao: He REALLY wants us to get an application | ||
+ | for PUF. This would be a great concrete application to sell. Because | ||
+ | the RNA enzyme cascade doesn’t just have to be with PHAT, it could | ||
+ | also be with any other pathway. And then we can also control when the | ||
+ | mRNA is expressed. | ||
+ | o Concerns: We need at least 3 different PUF binding sites and PUF | ||
+ | enzymes. And then we need to tether things to PUF. How possible is | ||
+ | this? | ||
+ | o Pan Silver has done this. Check up on it. (Slovenia 2010). | ||
+ | o We need to do a simple proof of concept: Make 2 PUFs. One that | ||
+ | inhibits GFP and one that inhibits YFP. And then after that make the | ||
+ | cascade. | ||
+ | o OR: we could do a simple 2 enzyme cascade. Ethanol is 2 enzymes. Is | ||
+ | there one that’s a color change? We can even use Slovenia’s reporter. | ||
+ | (Go find it). | ||
+ | Will fusion kill the binding activity of PUF? | ||
+ | o How do we figure out the spacing? It’s already figured out by Slovenia. | ||
+ | o We may want to add an artificial loop so it’s stiffer and the spacing is | ||
+ | easier. | ||
+ | o How difficult is it to tether onto PUF? | ||
+ | First get the Slovenia system, then address this. Figure out the | ||
+ | enzymes and then go from there. | ||
+ | Slovenia published a paper with Matthew Delisa (Cornell) to | ||
+ | produce resveratrol in E. coli via a DNA scaffold. | ||
+ | Also need to read the Pam Silver paper and Harvard paper. | ||
+ | - Should PHAT project keep looking into modeling how to make picetannol | ||
+ | more soluble? | ||
+ | o It would be difficult because it comes down to water molecules, which | ||
+ | aren’t great and are complicated because of the hydrogen bonding | ||
+ | networks. | ||
+ | o The calculations will be difficult. | ||
+ | o Not worth the time investment. | ||
+ | o Everyone should really be looking into the RNA scaffolding. | ||
+ | Characterizing | ||
+ | - Needed for silver medal | ||
+ | - Will be replicating Washington’s winning design (biodiesel) with the help of Dr. | ||
+ | Jin’s lab equipment. | ||
+ | - Look at different temps, different sugar concentrations, etc. | ||
+ | Moving forward | ||
+ | - Still need to prove PUF works | ||
+ | - Need to look into creating the mRNA backbone. | ||
+ | o Needs to be very stable! But since it’s non coding it’s naturally more | ||
+ | unstable. | ||
+ | o Normally stem loops will be sufficient to keep the ends safe. | ||
+ | o The stem loops that will dock the PUFs need to be oriented correctly. | ||
+ | They need to be pointing together, not apart. Can make scaffolds in | ||
+ | parallel and test them, but should start by using folding software. There | ||
+ | is RNA software that can predict hairpin structures, but not loops. And | ||
+ | they only give predictions. Look at mfold. It’s on a web server. It | ||
+ | doesn’t give one structure, it gives several and ranks them by free | ||
+ | energies. | ||
+ | Also consider introducing PUF binding sites in the middle of the | ||
+ | gene. | ||
+ | o Look into strains without RNA endonucleases. | ||
+ | - Find a 2 step color changing process to use as the cascade | ||
+ | o Could look into pigmented e. coli, but really should look at the Slovenia | ||
+ | project (even though they used zinc fingers instead of PUF and zinc | ||
+ | fingers are much bigger). | ||
+ | - Still work on PHAT, it’s our ‘future’ application. | ||
+ | - If we get all that done, then put the PHAT project on the RNA scaffold. | ||
+ | - Finish characterizing the Washington biofuel project. | ||
+ | Future questions | ||
+ | - Does PUF work in E. coli? It’s expressed but does it bind? | ||
+ | - What 2 enzyme system will we use? Does it work in E. Coli without PUF? | ||
+ | Does it work when tethered to PUF? (Hopefully will be color change). | ||
+ | - Have other iGEM teams done mRNA scaffolding? | ||
+ | How is the old work division changing? | ||
+ | - PUF proof of concept: We can make it simpler, and just do as much as | ||
+ | necessary. Don’t worry about making the whole repression system. Just do | ||
+ | with YFP for yes or no. Angela and Isiah will continue with the proof of | ||
+ | concept. | ||
+ | - Uros, Asha, Anthony, and Adi will begin working on the RNA scaffolding. | ||
+ | - PHAT group: Will take yeast biobricks and put them in E. Coli. | ||
+ | Everyone’s Homework: | ||
+ | - Read up on iGEM 2010 Slovenia | ||
+ | - Pamela Silver Paper about RNA scaffolds. (is from Harvard) | ||
+ | - By next Friday, have constructs done. | ||
+ | THE NEW WORKING PLAN | ||
+ | - Biobrick PUF | ||
+ | o YFP proof of concept. The control for this has the same number of | ||
+ | base pairs between the RBS and the YFP gene. | ||
+ | o Biobrick wild type PUF and PUF with endonuclease | ||
+ | - Find a 2 enzyme system that is color changing. | ||
+ | o Make sure it works in E. coli. | ||
+ | o Make sure it works when it’s attached to PUF. | ||
+ | o Look for the simplest possible system! | ||
+ | - RNA scaffolding | ||
+ | o Design the RNA scaffold. | ||
+ | o Run it on mfold. | ||
+ | o Design regulation mechanisms so that we can see it’s better than | ||
+ | DNA. | ||
+ | - PHAT project | ||
+ | o Clone DM3’s into E. Coli | ||
+ | o Biobrick TAL, 4CL, STS, DM3’s for e. coli | ||
+ | o Make the operon that will work in E. Coli | ||
+ | o If time permits: Bind the resveratrol pathway to PUF to use in the | ||
+ | assembly line. | ||
+ | - Characterize Washington’s Biofuel project. | ||
+ | o Qualifies us for silver. | ||
+ | </textarea> | ||
</div> | </div> | ||
<div id="meet22" style="display:none"> | <div id="meet22" style="display:none"> | ||
+ | <textarea rows="30" wrap="soft"> | ||
+ | </textarea> | ||
</div> | </div> | ||
<div id="meet23" style="display:none"> | <div id="meet23" style="display:none"> | ||
+ | <textarea rows="30" wrap="soft"> | ||
+ | </textarea> | ||
</div> | </div> | ||
<div id="meet24" style="display:none"> | <div id="meet24" style="display:none"> | ||
+ | <textarea rows="30" wrap="soft"> | ||
+ | </textarea> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 21:55, 19 June 2012