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Team:UIUC-Illinois/Notebook/MeetingNotes
From 2012.igem.org
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<div id="meetingselection1" class="meetingselection"> | <div id="meetingselection1" class="meetingselection"> | ||
| - | <center><h2>Pre- | + | <center><h2>Pre-April</h2></center> |
<li><a name="meet1">2/17/12</a></li> | <li><a name="meet1">2/17/12</a></li> | ||
<li><a name="meet2" >2/19/12</a></li> | <li><a name="meet2" >2/19/12</a></li> | ||
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<li><a name="meet8" >3/25/12</a></li> | <li><a name="meet8" >3/25/12</a></li> | ||
<li><a name="meet9" >3/27/12</a></li> | <li><a name="meet9" >3/27/12</a></li> | ||
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</div> | </div> | ||
<div id="meetingselection2" class="meetingselection"> | <div id="meetingselection2" class="meetingselection"> | ||
| - | <center><h2> | + | <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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<li><a name="meet17" >4/22/12</a></li> | <li><a name="meet17" >4/22/12</a></li> | ||
<li><a name="meet18" >4/24/12</a></li> | <li><a name="meet18" >4/24/12</a></li> | ||
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</div> | </div> | ||
<div id="meetingselection3" class="meetingselection"> | <div id="meetingselection3" class="meetingselection"> | ||
| - | <center><h2> | + | <center><h2>May-July</h2></center> |
| - | <li><a name=" | + | <li><a name="meet18a">5/1/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet19" >5/25/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet20" >5/29/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet21" >6/1/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet22" >6/11/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet23" >6/12/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet24" >6/18/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet25" >6/19/12</a></li> |
| + | <li><a name="meet26" >6/20/12</a></li> | ||
| + | <li><a name="meet27" >6/21/12</a></li> | ||
| + | <li><a name=""></a></li> | ||
| + | <li><a name=""></a></li> | ||
</div> | </div> | ||
<div id="meetingselection4" class="meetingselection"> | <div id="meetingselection4" class="meetingselection"> | ||
<center><h2>July-August</h2></center> | <center><h2>July-August</h2></center> | ||
| - | <li><a name=" | + | |
| - | <li><a name=" | + | <li><a name="meet28" >7/5/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet29" >7/6/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet30" >7/9/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet31" >7/10/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet32" >7/11/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet33" >7/12/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet34" >7/16/12</a></li> |
| + | <li><a name="meet35" >7/17/12</a></li> | ||
| + | <li><a name="meet36" >7/19/12</a></li> | ||
| + | <li><a name="meet37" >7/20/12</a></li> | ||
| + | <li><a name="meet38" >7/25/12</a></li> | ||
| + | <li><a name="meet39" >7/30/12</a></li> | ||
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</div> | </div> | ||
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<div id="meetingselection5" class="meetingselection"> | <div id="meetingselection5" class="meetingselection"> | ||
<center><h2>August-Jamboree</h2></center> | <center><h2>August-Jamboree</h2></center> | ||
| - | <li><a name=" | + | |
| - | <li><a name=" | + | <li><a name="meet40" >8/6/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet41" >8/10/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet42" >8/24/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet43" >8/31/12</a></li> |
| - | <li><a name=" | + | <li><a name="meet44" >9/15/12</a></li> |
| - | <li><a name=" | + | <li><a name=""></a></li> |
| - | <li><a name=" | + | <li><a name=""></a></li> |
| + | <li><a name=""></a></li> | ||
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</div> | </div> | ||
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<div id="meet1" style="display:none"> | <div id="meet1" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM advisors meeting | iGEM advisors meeting | ||
2/17/12 Meeting | 2/17/12 Meeting | ||
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<div id="meet2" style="display:none"> | <div id="meet2" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Weekly Meeting – Sunday, February 19, 2012 | iGEM Weekly Meeting – Sunday, February 19, 2012 | ||
Getting card access to IGB | Getting card access to IGB | ||
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</div> | </div> | ||
<div id="meet3" style="display:none"> | <div id="meet3" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM meeting minutes from 2/26/12 | iGEM meeting minutes from 2/26/12 | ||
- We watched the first 13:00 min of this video about Imperial College of London | - We watched the first 13:00 min of this video about Imperial College of London | ||
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</div> | </div> | ||
<div id="meet4" style="display:none"> | <div id="meet4" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Meeting Minutes | iGEM Meeting Minutes | ||
3/4/12 | 3/4/12 | ||
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</div> | </div> | ||
<div id="meet5" style="display:none"> | <div id="meet5" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
Agenda of Advisor Meeting | Agenda of Advisor Meeting | ||
3/6/2012 | 3/6/2012 | ||
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</div> | </div> | ||
<div id="meet6" style="display:none"> | <div id="meet6" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
3/7/12 Engineering council | 3/7/12 Engineering council | ||
Present: Asha, Cara | Present: Asha, Cara | ||
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</div> | </div> | ||
<div id="meet7" style="display:none"> | <div id="meet7" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Meeting Minutes | iGEM Meeting Minutes | ||
3/11/12 | 3/11/12 | ||
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</div> | </div> | ||
<div id="meet8" style="display:none"> | <div id="meet8" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Meeting Minutes | iGEM Meeting Minutes | ||
3/25/12 | 3/25/12 | ||
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</div> | </div> | ||
<div id="meet9" style="display:none"> | <div id="meet9" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM advisor’s meeting | iGEM advisor’s meeting | ||
3/27/12 | 3/27/12 | ||
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</div> | </div> | ||
<div id="meet10" style="display:none"> | <div id="meet10" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Weekly meeting | iGEM Weekly meeting | ||
4/1/12 | 4/1/12 | ||
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</div> | </div> | ||
<div id="meet11" style="display:none"> | <div id="meet11" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Advisor’s meeting | iGEM Advisor’s meeting | ||
4/3/12 | 4/3/12 | ||
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</div> | </div> | ||
<div id="meet12" style="display:none"> | <div id="meet12" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Engineering Council | iGEM Engineering Council | ||
Elections | Elections | ||
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</div> | </div> | ||
<div id="meet13" style="display:none"> | <div id="meet13" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM weekly meeting | iGEM weekly meeting | ||
4/8/12 | 4/8/12 | ||
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</div> | </div> | ||
<div id="meet14" style="display:none"> | <div id="meet14" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Saturday meeting | iGEM Saturday meeting | ||
4/14/12 | 4/14/12 | ||
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</div> | </div> | ||
<div id="meet15" style="display:none"> | <div id="meet15" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM weekly meeting | iGEM weekly meeting | ||
4/15/12 | 4/15/12 | ||
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</div> | </div> | ||
<div id="meet16" style="display:none"> | <div id="meet16" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Advisor’s Meeting | iGEM Advisor’s Meeting | ||
4/17/12 | 4/17/12 | ||
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</div> | </div> | ||
<div id="meet17" style="display:none"> | <div id="meet17" style="display:none"> | ||
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iGEM Weekly Meeting Minutes | iGEM Weekly Meeting Minutes | ||
4/22/12 | 4/22/12 | ||
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</div> | </div> | ||
<div id="meet18" style="display:none"> | <div id="meet18" style="display:none"> | ||
| - | <textarea rows="30" wrap="soft"> | + | <textarea readonly rows="30" wrap="soft"> |
iGEM Advisor’s Meeting | iGEM Advisor’s Meeting | ||
4/24/12 | 4/24/12 | ||
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reading day’s social outing.</textarea> | reading day’s social outing.</textarea> | ||
</div> | </div> | ||
| + | |||
| + | <div id="meet18a" style="display:none"> | ||
| + | <textarea readnoly rows="30" wrap="soft"> | ||
| + | iGEM Advisors Meeting | ||
| + | 5/1/12 | ||
| + | |||
| + | Announcements | ||
| + | Courtney: $500 is needed for travel funds because of a new fee that requires $25 per ticket, in addition to the ticket price. We will continue looking at departments and companies for funding, but all meetings to request funding should be set up with Courtney present. We should also contact companies in research park (Adi will take the lead on this). Our goal is to recruit $10,000 from various departments and companies in order to be financially secure this year and set up next year’s team. Adi will contact the ACES department (through Bhalerao) and Steven Sliger of MCB. | ||
| + | |||
| + | Agenda | ||
| + | Wetlab Summer Plans | ||
| + | PUF | ||
| + | Make multiple PUF biobricks out of existing PUM1 genes and PUM1+endonuclease domain (provided by Dr. Wang at UNC) | ||
| + | 3 ways to test is PUF really binds with the construct | ||
| + | Path 1: Put PUF binding site between the RBS and YFP. If no YFP is detected then PUF has successfully bound. We can also test various PUF binding sites: before the RBS, after the YFP, try using multiple PUF binding sites before the RBS | ||
| + | Suggestion: put the endonuclease site inbetween the RBS and YFP so that when PUF binds we separate the RBS from the reading frame. | ||
| + | Controls: In the positive control, put a PUF binding site, but don’t express PUF. That way we can see that the PUF binding site doesn’t disrupt the YFP expression on it’s own. | ||
| + | Recommendation: This is the immediate and primary focus. | ||
| + | Path 2: PUF between RBS and LacI. If PUF binds then LacI is repressed. The LacI is controlling the YFP, so we now see a modular response to PUF binding. We can also test the different locations of the PUF binding site within this model. | ||
| + | Path 3: UNC has an incomplete project working with a PUF library that recognizes certain genes. We would use the PUF with the endonuclease domain to cut the designated genes. The genes from this library can get made into biobricks. | ||
| + | Details and illustrations of this plan are included in the handout (attached to the meeting minutes). | ||
| + | How are we selling the PUF project? Why are we doing this? Why do people care? | ||
| + | We are creating a research toolkit that uses PUF and linked proteins for translational repression. | ||
| + | This is another technique used to make a gene circuit, but can also be further used in therapeutics, such as treating HIV of myotonic dystrophy. | ||
| + | This puts us in the “new application” category. | ||
| + | It is the hope that this ends up being more effective/faster/cheaper/specific, etc. | ||
| + | We want to sell this project to the judges (both for iGEM and entrepreneurial). | ||
| + | We need to find a current project so we have a specific, concrete example of an awesome application. This is key to market ourselves. We need to show that we have helped solve a problem because PUF was better than any other option available. | ||
| + | 1st step in the entrepreneurship project is to look at patents and trademarks for already existing methods of translational repression. | ||
| + | We need to have a specific need and market to satisfy. Specific examples are key! Data is also good. | ||
| + | Do the simple key wetlab parts of this project, and continually research applications during the summer. | ||
| + | Resveratrol/Piceatannol | ||
| + | Korean lab under Prof. Chul-Ho Yun is willing to share mutant strains with us, and the first author of the paper, Donghyun Kim, is a post-doc in the Kemper lab on campus. Cara is in communication with both the lab and the author | ||
| + | ChemSketch is being used to draw derivatives of piceatannol and then calculate the logP value of that compound to find the solubility. Then use the Molecular operating environment software to model docking of these more soluble compounds into the insulin receptor. Then would turn at least one of the mutant strains (BM3) from the Korean lab into a biobrick. | ||
| + | Resources: The school of Chemical sciences is being contacted about using their software, and the Hergenrother, Spies, Nair, and Cobb labs are also being contacted. 1-2 other iGEMmers will work with Cara on this project. | ||
| + | Recommendations: In vitro production of piceatannol is expensive and time consuming (purify enzyme, etc). But if we pursue a fermentation, we can produce large amounts of piceatannol, which is a very good application and great for our entrepreneurship competition. We could even produce our own reseveratrol to feed into the piceatannol fermentation. Potential problem: can we secrete piceatannol? | ||
| + | Discussion | ||
| + | The PUF project is technique based and the piceatannol project is application based. It’s great that we have both of them and they should be pursued in tandem. | ||
| + | Proposed budget: asking for $21,000 in materials and supplies, but we can get many free supplies. | ||
| + | Don’t worry about money, if we come up with a good wetlab project, then it becomes easier to obtain funding to travel to the actual competition. | ||
| + | We have enough money to do both projects in tandem. | ||
| + | Call Amanda about GeneArt. We may have money to use up with them. | ||
| + | If we check with Courtney before purchasing anything, we should be fine with money. | ||
| + | Once we start doing work, it’s easier to get funding. This is the big idea. | ||
| + | Our budget this year is actually bigger than normal, so as long as we work hard and are responsible, everything should work out. | ||
| + | |||
| + | Voting on Workload | ||
| + | PUF: Angela, Isiah, Uros, Anthony, Adi *, Asha * | ||
| + | Anthony and Isiah, Uros and Asha, Angela and Adi | ||
| + | Phat: Cara, Divya, Bob * | ||
| + | * = have other primary responsibilities within iGEM. Adi is running entrepreneurship, Bob is running the wiki, Asha is running human practices. | ||
| + | Social Event | ||
| + | This Friday, Golden Harbor. Cara will attempt to make reservations. We will meet at Engineering hall at 5:30 to leave on the 5:40 Green. Please be on time! =) | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
<div id="meet19" style="display:none"> | <div id="meet19" style="display:none"> | ||
| + | <textarea readonly 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 readonly 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 readonly 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 readonly rows="30" wrap="soft"> | ||
| + | Monday, 6/11/12 | ||
| + | - Divya, Washington characterization – transformed the petrobrick into the K 12 plasmid. The petrobrick uses ADC and ADR to make biofuel. | ||
| + | - Angela and Anthony can’t make it to high school jamboree | ||
| + | - Asha, human practices – high school jamboree is overnight on Friday, June 29 and all day on Jun 30th. Let me know ASAP if you can’t go. Also, for the GMO video we are now researching transgenic plants | ||
| + | - Bob, webmaster – is open to suggestions about progress on the wiki, need content and progress on the projects so that companies can see our progress, primers/structures/data etc should be sent to Bob so that it can be included in the wiki (it doesn’t have to be fancy), will look into putting a pretzi presentation on our wiki so that we can have an interactive flowchart element | ||
| + | - Adi, corporate – will have the elevator pitch done in time to present it at Friday’s advisor’s meeting, sent an email with supplies to Gold Biotech | ||
| + | - Cara, PHAT – transformed, emailed Parts registry and said that the parts will be shipped by the end of the week (we can expect them around next week) | ||
| + | - Uros – transformed split GFP (with Asha), has a rough draft of an RNA scaffold (will get put in the dropbox), got a program to work and is experimenting with placement of the PUF binding site, it’s looking to be 123 base pairs so it’s possible to synthesize it, is still looking to contact the Pam Silver author and RNA contacts on campus, will also further look into the crystal structure of binding PUF, hope to have a final draft of the RNA for Friday | ||
| + | - Angela – this is a busy week for many advisors so simply go to their offices and chat with them, Courtney is leaving in a few weeks so if we need to order anything let’s try to do that now, will teach Isiah how to do an order form, will be taking a leave starting on Friday to take care of her mother in Taiwan. | ||
| + | |||
| + | </textarea> | ||
</div> | </div> | ||
<div id="meet23" style="display:none"> | <div id="meet23" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Tuesday, 6/12/12 | ||
| + | - Bob, webmaster - Adi has added a basic pretzi to the wiki, google issues when searching for our wiki (need to get rid of old pages) | ||
| + | - Divya, publicity - has created a logo and sent it to Bob, worked on blog | ||
| + | - Washington characterization - cells grew, they aren’t being fed sugar so a control group of the petrobrick without sugar (so now alkanes), will talk to Jin about media and GCSM analysis | ||
| + | - Cara, PHAT - grew 2 cultures of each strain of BM3 for glycerol stock and miniprepping tomorrow, got Falcon tubes from MMG | ||
| + | - Uros, RNA scaffold - got a haircut, talked to Todd and got PUF binding sites, it seems that a good plan would be to sythesize it and put it in a plasmid. IGT has a 500 base pair synthesizing deal for $100. Todd will be giving us one of the plasmids, we already have the other one. The duet has 2 promoters so it can have 2 genes. One of the promoters will be used for the split GFP. In the future we will need 2 duets. One will encode the plasmid and then there is room for one other gene. That means we can get up to 3 genes for picetannol production. | ||
| + | - Asha - worked on human practices stuff, got 3 boex for the -80. One box has the electrocompetent cells, and the other boxes are for PUF/PHAT as needed. Also inoculated both split GFPs and J04500. | ||
| + | - Angela, PUF - we do have the plasmid with the testing part (PUF binding site and control), but are waiting on primers for the PUF gene so we can subclone it. Transform the 2 plasmids into BL21 tomorrow for testing. AFter the completion of this control, we will do as many PUF constructs as possible as a backup plan for the scaffolding. | ||
| + | - | ||
| + | |||
| + | </textarea> | ||
</div> | </div> | ||
<div id="meet24" style="display:none"> | <div id="meet24" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Monday, 6/18/12 | ||
| + | - Adi, corporate - contacted IDT and VWR, tried to contact Fisher. Need to send Courtney a list of things to buy and also forward the email that was sent to Gold Biotech. Will upload a corporate folder to Dropbox. This will help us create a list of companies and their statuses on funding. | ||
| + | - Elevator pitch: (this will be in video form later... also, team/company name is IllinoisSynth, product is IVOPC - in vivo o.. picetannol cascade). Edit info about team. Elaborate on obesity epidemic facts. Take out part about standardized tool kit, because that is no longer our product. Can discuss our process though, how we moved from the toolkit to a concrete application. Otherwise good! See the full text on the iGEM facebook page. Depending on how much work actually gets done, this pitch will change slightly. | ||
| + | - Bob, webmaster - just updated the notebook section. Needed to put the actual iGEM logo, upright and at the top of the page, how does everyone like the format? What is the length of our scaffold? How many genes are on it? (Plan, Uros: 2 duet plasmids. One space will be for the scaffold itself. Cara: we can go either tyrosine -> resveratrol, p-cumaric acid-> picetannol. Both of these steps are 3 enzymes. But to go straight from tyrsoine -> picetannol is 6 enzymes... which is too many). | ||
| + | - Asha/Anthony, PUF - 3rd PCR failure, there are primer dimers with only the lightest bands at the 700 bp mark. Potential problems with primer concentration. Will redo those calculations at 8:30 tomorrow. | ||
| + | - Isiah/Divya, Washington characterization - Isiah will be working in the morning, Divya will be working in the afternoon. Running a gel for the petrobrick right now. Will make media tomorrow. Still several days away from needing the | ||
| + | - Cara, PHAT - need to get resveratrol/picetannol (will order and will get reimbursed), need a concentration in which to bathe the cells to produce picetannol (will start with in vitro concentration from the paper), performed TLC in lab today and feels confident to move forward with it for resveratrol → picetannol converstion. The orgo lab professor is willing to help and donate supplies. Parts from the registry have not yet been received, should have come in today and will check again tomorrow (the part is the 2 ligated genes from yeast). | ||
| + | - Uros - RNA sequence has gotten final approval from Todd and Courtney, the ordering is dependent on IDT funds, emailed Melissa about it but she’s out for the week, Adi emailed IDT about it. Will not submit the sequence until we know that we have money. Tomorrow will transform cells with both GFP contructs to see if there is florescence with no scaffold. The duet plasmid is no longer useful because the split GFP will be tethered to PUF and the cell can support 2 plasmids. Can it be done through Genscript? IDT is offering 40% off to iGEM teams. After the split GFP, will help out and look into how to tether the PUF to the split GFP and other enzymes. | ||
| + | |||
| + | </textarea> | ||
</div> | </div> | ||
| + | <div id="meet25" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | |||
| + | Tuesday, 6/19/12 | ||
| + | - Anthony, PUF - calculated primer concentrations | ||
| + | - Asha, PUF - inoculated E0030 which is the correct PCR template, skyped with Angela to clarify stuff | ||
| + | - Divya, Isiah, Bob, Washington characterization - ran a gel to check the inoculation of the petrobrick and it appears to be the correct thing (5 kb plasmid backbone), tomorrow will go talk to Jin about part inconsistencies, Isiah and Divya will make the media tomorrow in the morning | ||
| + | - Bob - helped Washington characterization, used the extra gel in the ice cream room, got the part with resveratrol and TAL, then plated it. Will check it in the morning and inoculate it tomorrow afternoon. for the wiki, finished putting up all the meeting minutes | ||
| + | - Uros - need to insert both split GFP into a duet plasmid, because otherwise the plasmids will compete. Designed primers to insert restriction sites to the split GFP. | ||
| + | - Need to update the daily work summaries. | ||
| + | |||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | <div id="meet26" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | |||
| + | Wednesday, 6/20/12 | ||
| + | - Adi, corporate - revising the elevator pitch, working on the executive summary, going to carry out market research from a primary perspective, which will involve emailing a survey to companies. Weekend of July 6, will go to Chicago to meet Alex and finish the entrepreneurship website. Made excel sheet on a google doc of companies | ||
| + | - Bob, Cara, PHAT - plated the 4CLSTS on 4 plates of different resistances. | ||
| + | - Bob, webmaster - Angela sent suggestions for the wiki, changed the header a little, emailed Bhalerao to get a remote service to work with the server | ||
| + | - Anthony - DCL access? work distribution is different. | ||
| + | - Asha, PUF - PCR worked! Need HindIII! Need to email Will about enzymes in the big -20 (Uros will do this). Will talk to Courtney about the enzymes and traveling to the jamboree tomorrow. We got SORF funding. Will come up with a human practices website for SORF. | ||
| + | Tshirt sizes: Adi (M), Bob (S), Uros (M), Asha (S), Cara (S), Divya (S), Isiah __, Angela (S) | ||
| + | - Divya, Isiah, Washington characterization - talked with Jin, he wants a presentation at the advisor’s meeting, so Divya has been working on it. After the presentation he will discuss more of the project. | ||
| + | - Cara - Dr. Metcalf is giving us a culture that has the last enzyme we need. After that will design primers and proceed to get it. Still trying to grow the yeast enzymes. Ordered the resveratrol and picetannol. Sent an email to Mark about the reimbursement (Melissa is handling it) Dr. Rafferty is talking to the Chem department about getting supplies for TLC. Brad suggested putting all of the enzymes in an operon under one promoter. Having some trouble with the agar stab that has the part. | ||
| + | - Uros - ordered the part, but then got word from IDT that they can’t make it because of the large stem loops. Apparently the G block synthesis isn’t well suited for stem loops and secondary RNA structures. The IDT guy suggested another method of synthesis, but it’s only up to 200 bp. We might need to choose only 1 scaffold. Will consult with Todd again tomorrow. | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | <div id="meet27" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | |||
| + | Thursday, 6/21/12 | ||
| + | - Adi, corporate - worked on executive summary, sent an email to Sarstedt (Courtney met their rep at a trade show), will send an email to NEB. | ||
| + | - Divya - worked at presentation on Washington characterization for Dr. Jin, will work on finalizing it with Isiah tonight and then will send it to Uros. It will be presented at the advisor’s meeting tomorrow. Will be adding more blog entries. | ||
| + | - Anthony - worked on GFP tethered to PUF stuff, will talk to a grad student tomorrow, is free after 1, but Todd is free after 10:30. Could also talk to Brad if he isn’t busy. Sequences are prepared, but need to adjust order and further edit it. Also have to figure out the actual manufacting synthesis (genscript money or PCR technique?) | ||
| + | - Asha - ran a digest of PUF binding, contacted Courtney and Melissa about the high school iGEM trip, GMO video script is being made, Uros will look into getting cameras from the art department | ||
| + | - Cara - got transfomed cells, will miniprep them, will sequence the part because it has been labeled as unreliable, need primers for it. Will order p cumaric acid (1 gram for $8). Will try to clone it and express it in e. coli. (p cumaric gets turned into resveratrol). Will get rotobactercapsiaus that has the enzyme to turn tyrosine into p cumaric acid. Will make some constructs for the advisors meeting tomorrow. | ||
| + | - Bob - got access to the server from Bhalerao. Needs to learn linux to work it (Ubuntu). Will work on human practices page with Asha. | ||
| + | - Uros - advisor’s meeting at 3 tomorrow. Everyone should try to create some sort of presenation. (Anthony won’t be able to make it). Send presenations to Uros. miniprepped and made a glycerol stock of the pETDuet-1 from Todd. Also digested the first construct for it and will ligate it tomorrow. Also talked to Todd about synthesizing the RNA scaffold. A promising option is making a ‘mini-gene’ but it takes 2-4 days just to get a quote. Emailed Dr. Pamela Silver questions about using IDT as a synthesis (they did it without G block synthesis, so probably used the mini-gene option.) If correspondence goes well, then hopefully we can get her to look at our sequence. It might be more expensive than anticipated, but we’ll see. Also working with Anthony on how to tether split GFP to PUF. will flip one of the PUF binding sites in order to put the split GFP closer together. | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | <div id="meet28" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | |||
| + | Thursday, 7/5/12 | ||
| + | - Adi, corporate/entrepreneurship - Been working with Alex on the entrepreneurship wiki, went to Chicago to do design the website with Alex. At this point have the entire structure of the wiki, but are hosting it on a different domain so that the code can get perfected and then uploaded to the iGEM wiki. So far, it looks so good. The iGEM entrepreneurship wiki should be up in around 2 weeks. Once there is data it will get uploaded and the entrepreneurship code can get transferred to the normal wiki if necessary. Also, obtain $250 from the Roy J. Carver Biotechnology Center, Courtney has given the approval to use this to buy enzymes. SARSTEDT is also going to sponsor us, but they have not specified an amount yet. | ||
| + | - Anthony, PUF biobricking - looked at gel of PSB1C3 run by Asha. The bands were not correct, so digested more. Each had 2 bands and none of them were the right size. Will use linearized plasmid next. Made LB and autoclaved more 1.5 centrifuge tubes. | ||
| + | - Asha, PUF - still waiting on WT PUF/ pBAD primers, so in the meantime I made 11 tubes of DH5a cells. And plated YFP constructs from glycerol stock to do colony PCR again. | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | <div id="meet29" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | |||
| + | Friday, 7/6/12 | ||
| + | - Adi, Entrepreneurship - same as yesterday. entrepreneurship wiki, money from Roy J. Carver (for sequencing), some sort of sponsorship from sarstedt. DWR and Fisher haven’t really responded, so Courtney will contact them personally to try to get a response. Will contact Ginkgo Bioworks next. Will also set up times outside of advisor’s meetings to meet with Joe Bradley. | ||
| + | - Anthony, PUF - PCRed 2 tubes of WT PUF, started 3 ligations with 3 different digestions and the one type of PSB1C3 that kind of worked. After the biobricking, will help Angela and Asha with the PUF and pBAD. | ||
| + | - Isiah, Washington characterization - Don’t have to wait for alkanes, so on Monday when Divya returns they will start the media growth along with the K12 and Petrobrick. Ideally by the end of the week alkanes should be produced and that fact means the petrobrick was successfully characterized. Need filters for 500 mL of liquid. Actually would be ideal to start media growth earlier, so will talk to Divya about that. Courtney has trained Isiah on how to order things, so when she leaves we will order things through Isiah (Courtney will still approve purchases remotely). | ||
| + | - Asha, PUF - Did colony PCR on the YFP constructs, PCRed WT PUF for pBAD, ran the gel for both and saw primer dimers for all. Must redo. Using the right primers? Reused the old YFP primers as check primers, which should work but might not. Will look for the check primers. Human practices: get mike from the Art department (connected to the museum) open from 11-5 during the summer (go to the second floor and talk to them). | ||
| + | - Uros, RNA scaffolding - continued work on split GFP. Had to redo a gel so ran the digestion for 4 hours (an extra hour from last time). Did a gel on it, and will continue ligation. The split GFP should be done and sent out for sequencing on Tuesday. Next Friday is the goal for tethering the split GFP to PUF. thinking about characterizing the scaffold, but putting that off until the split GFP is done. Will need assistance with characterizing the split GFP and scaffold. Will characterize the scaffold by RTPCR (being trained currently). Will consult Pam Silver paper on how they did it (think they did microscopy, but that might not be the best option for us). | ||
| + | - Angela - We are on track according to the 2012 iGEM judging form: characterization (gold), human practices (gold), working on biobricking and characterization of PUF (silver). The sequencing and characterization of PUF will happen in parallel to save time. A deadline for finishing characterization is next Friday (7/13). That way by the end of July we will be meeting all the qualifications for the gold medal. This is a very strict deadline because we have less than 2 months left to do lab work. The regional jamboree is the first week of October, and September is completely dedicated to perfecting the poster and presentation. We are running out of time, so everyone needs to work as hard as possible. Angela, Asha, and Anthony are all working on the same PUF plan to try to finish the PUF and YFP cloning done by Monday. Then we can begin the characterization and hopefully finish that by Friday. | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | <div id="meet30" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Monday, 7/9/12 | ||
| + | - Adi - meeting with Joe this week to look over entrepreneurship executive summary, are being sponsored by SARSTEDT (box of supplies came today), emailed thank yous to sarstedt and gold biotech. Should contact Octochem about further sponsorship. | ||
| + | - Divya, Washington characterization - did the M9 media for alkane production, for publicity is writing an email to IMSA, Asha took pics for blog, Divya will email Flickr password out. Will talk to Isiah about tshirts. After actually obtaining alkanes, will write up the characterization with isiah. Followed a bunch of people on twitter (37), and currently have 20 followers. Also need to make a new trifold for Quad day and E-night. Add any other publicity ideas to the google doc. | ||
| + | - Cara, PHAT - have designed sequencing primers but not ordered them yet (wants to try using VR and VF), might need to design another set of sequencing primers to account for 100 bp mess up at the start of every sequence. UPenn is going to ship genetic info on rotobacter at sometime next week because the rotobacter from Dr. Metcalf isn’t working. Is talking to someone in MMG about high performance thin plate chromatography. Have glycerol stocks of BM3s in expression vector. Get things characterized before biobricking (plus, biobricking primers aren’t in right now). Would like to buy malonyl-CoA for $80, but haven’t talked to Courtney about it yet. | ||
| + | - Bob, webmaster - redoing PCR for 4CL:STS and will run gel later, wiki will be changed so that the lab notebook is separated among the PUF and PHAT projects. Have bullet points and pictures, doesn’t need to be very intense. Keep it simple and clear. Each day has a data section with numbers or pictures divided into the PUF and PHAT projects. Can also have tabs and drop down menus. Also uploaded most recent minutes. Will also add Sarstedt to the sponsors page. | ||
| + | - Anthony, PUF - did all steps from PCR to transformation of WT PUF. The transformation didnt work so have started redoing it. | ||
| + | - Asha, PUF - working on re-doing the eYFP in protet constructs (both the control binding site and the PUF binding site.) Did PCR today, and needs to run the gel later. Then will religate, transform, grow, do colony PCR, send to sequencing and begin characterization. Eventually, will clone in the WT PUF in pBAD to the cells. Also making plate (10 LB, 25 Amp, 25 Amp and Cm) and making more pBAD. Will contact local science camps about presentations/assisting in lab for outreach. | ||
| + | - Angela - after doing digestion, you don’t always have to do gel purification, can just do PCR cleanup. Because alot of these runs are low copy number, so gel purification yields low numbers. Did tons of PCR for PUF and most worked. No pBAD worked in the digestion, so got more pBAD from Asha and will hopefully have finished digestion/ligation/transformation by tomorrow. Will then send in for sequencing. | ||
| + | - Uros - trying to get the pETDUET to work with split GFP, but hasn’t really worked. the pETDUET plasmid is low copy number and so we have low yield and have a really faint band with split GFP. If we sent the split GFP tethered to PUF to sequencing on Friday, we would get it at the end of July. It’s expensive and takes a while but we know that we would have it. If we did it by ourselves it would be difficult, and take an undetermined amount of time. | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet31" style="display:none"> | ||
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| + | Tuesday, 7/10/12 | ||
| + | - Divya, washington characterization - still waiting for the stuff to grow, will email Isiah about further updates, need to talk to Jin about GCMS on Thursday. helped Asha plate some stuff, will make plates tomorrow. | ||
| + | - Anthony - looked a PCR gel. only 1 of 3 worked, but it was a lot of yield, so it will last a couple of rounds of digestion/ligation. So the digest/ligate will occur today and be finished tomorrow. | ||
| + | - Asha -YFP PCR worked. looked at other YFP colonies under UV light and none of them glowed =( so it’s a good thing we’re re-ligating! ruined 70 plates... sorry! =( | ||
| + | - Angela - did ligation/transformation and will see if it worked later tonight. designing primers for primer extension (backup plan). Being trained in Rao’s lab to use sewing and use the florescent meter to measure the mcherry and venus for the reporter genes. Asking to be trained for mathematical modeling for the output of our project. | ||
| + | - Uros, scaffold and tethering - working to tether PUF and GFP. Read the UNC paper (draft) on how they tethered, but will need more info because there is no supplemental info or data. Also need to send some emails out and continue reading up on it. Wants to talk to Todd about tethering and the low copy plasmid. Ligations didnt work and transformations didnt work =( | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet32" style="display:none"> | ||
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| + | Wednesday, 7/11/12 | ||
| + | - Asha, PUF - yesterday digested eYFP and protet and got bands of the correct length. Ran ligation for 7 hours using a 10:1 ratio of insert:vector. Transformed today, Bob and Cara plated for me. Will look at plates tomorrow. If colonies, will look at them under the UV light to see if they glow! | ||
| + | - Divya, made CM, Amp, and Amp+CM plates for Asha, talked to Jin about GCSM: work with Quan Lee (?), Isiah will pick up alkane production tonight, will resuspend tomorrow in EtOAc to extract | ||
| + | -Bob, put Starssted on the sponsor’s section, and worked | ||
| + | -Angela: | ||
| + | Constructed primers using primer extension method for the entire PUF project constructs. Primers for cloning of YFP to ProTet and wild type PUF to pBAD. To be more precise the method is termed CPCA used for cloning. The benefits are that it doesn’t need any ligation or gel purification. Once the primers arrive, probably Friday, we can try the PCR and check whether the insert is present or not by check primers. Check primers were ordered today as well. Wild type PUF need another set of sequencing primers. Orders have actually not been placed yet, it depends on Courtney. CPAC method doesn’t leave any scars in the cloning and no gel purification is required, only PCR and gel running is required. Perhaps the method will be taught sometime later in the week to the whole group. One modification is that instead of using E0030, Venus will be used. PSB1C3 will be arriving on Friday. Transformation after ligation was done today, colonies will be seen tomorrow. Starting another set of regular cloning in case CPAC doesn’t work, from digestion/ligation/transformation. | ||
| + | -Adi: | ||
| + | Alex will be arriving this weekend. The entrepreneurship wiki should be done in approximately 2 weeks. Adi will e-mail Mark from Octochem about a chemical needed for the PHAT project. | ||
| + | -Anthony: | ||
| + | Digested, extracted, and ligated PCR of pBAD and wild type PUF PCR. Transformed today and will plate, hopefully there will be colonies tomorrow. | ||
| + | -Uros: | ||
| + | Continued to investigate how to tether PUF to a correct split-GFP sequence. Asked Angela for further information on the UNC paper in order to receive more information where on PUF was the tether placed. Proceeded to e-mail the graduate student who helped collaborate and give advice for the RNA Scaffold synthesis about the split-GFP sequence used in the paper (Pam Silver paper). E-mailed Dr. Bourde, the principal investigator of the lab which was referenced in the Pam Silver paper, concerning split-GFP work for either the sequence or possible part shipment. E-mailed Todd in order to get more information or suggestions on where to head with the tethering project. Also, Todd might offer insight whether it is still feasible to produce the construct ourselves before August and so we could save money before synthesis. Been trying to find a trustworthy source for the split-GFP sequence and have found two sources. One of them being from a past iGEM team and the other from a research article. For Friday an update will be presented to the advisors along with a list of options which can be pursued, one of which will be decided upon collectively. An e-mail was forwarded from Courtney, sent by IDT, that the RNA scaffold gene is still in production and everything is being done so it could be properly made. Any other product ordered along with the scaffold should be asked to be sent immediately due to the unpredictable completion time of it. | ||
| + | -Cara/Bob: | ||
| + | Cara and Bob made smaller aliquots of freshly made LB media and put them into the 50mL conical tubes. The contents of the conical tubes should be transferred into small glass containers and the tubes autoclaved/reused. Gloves arrived today, needed materials need to be updated and immediately communicated to Courtney. Rafferdy got back to Cara and has TLC plates for her to use. TLC standards worked yesterday, solvent used was 85:15:3 chloroform, methanol, acetic acid. Reverse phase was tried as well. 10mL of solvent was used, 250mL of solvent was made which should last a while. All reagents were received from MMG. Gels were ran to check whether PCR worked last night, PCR might have worked. The gels need to be extracted and sequenced in order to check whether it worked. IDT will be e-mailed/called to separate the orders of scaffold and primers due to delay of the scaffold production. 5mg of piceattanol and 100mg of resveratrol are in the inventory. All piceattanol contents were resuspended in ethanol. Resveratrol will be diluted as well in some solvent. Starting fermentation is the next step. Bob plated some things for Asha after transformation. Sponsors were also added to the Wiki and the lab notebook section was updated. Meeting with Melissa will be held by the end of the week. WashU credited UIUC coding done by Bob for the Wiki!! | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet33" style="display:none"> | ||
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| + | Thursday, 7/12/12 | ||
| + | - Divya - Isiah got the cultures out. Talked to Jin and it turns out that we actually need to wait until the alkanes arrive to use them as a standard for GCMS. Talked to the GCMS lady about conditions. Divya will look up the info and email it to the lady to create the standard curve. Either tomorrow or monday results will occur. On next Monday, will begin helping with cloning. | ||
| + | - Asha - transformation of YFP ligation didn’t work, so replated. Also another ligation with only a 5:1 insert:vector ratio. Also went to the art department to ask about filming equipment for human practices. The checkout counter wasn’t open, but got an email to contact. | ||
| + | - Anthony - failed transformation from yesterday so replated. Also transformed again with a different ligation. Among 4 plates hopefully there will be results! =) | ||
| + | - Bob - ran a gel of Cara’s PHAT optimized PCR. Started a digestion of it. Working on wiki, more specifically putting all of the lab notes from the google doc on the wiki in nice little tables. Will also contact Melissa. | ||
| + | - Cara - did gel purification of the PHAT optimized PCR. The band is not the right length, but the PCR has consistently yielded this band. Therefore purified it and wants to find what it is. The suspicion is that the primers and PCR are working and the part is messed up. Digesting the vector or sequencing the part are good things to do at this point. Will type up a protocol from Brad and send it to everyone about growing things in a controlled environment for PHAT. | ||
| + | - Uros - talked to Todd in detail about the tethered PUF to CFP construct ourselves. He thinks that we can do it ourselves and complete it before August. The plan involves making alot of primers (angela will help). In parallel will construct a plasmid with the CFP parts so that we can have a control to see how the CFP works. have to check if CFP is in the kit and if we can just PCR it. Still the debate: do we synthesize or do it ourselves? it’s the problem of time and money. will sit down and create a very detailed plan with Todd tomorrow. | ||
| + | - Adi - been working on the wiki, Alex will come down on Saturday and everyone should come to lunch to meet him and discuss | ||
| + | - Angela - everyone needs to create a powerpoint of work and data to present at the advisor’s meeting tomorrow. Having problems with comp cells because transformations aren’t working. So is going to take some iGEM comp cells and try the same ligation transformations. It that doesn’t work will then transform with boughten comp cells. Prepared more ligations so will continuously ligate and transform until results. Finished the primers for primer extension and checked with Kori about making a standard protocol. Next week the primers will arrive and will start cloning the modular PUF reporters via primer extension. | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet34" style="display:none"> | ||
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| + | 7/16/12 Monday | ||
| + | Reflection: | ||
| + | |||
| + | Optimistic goals: | ||
| + | *By the end of the summer, 4CLSTS and BM3 and mutants BM3 tethered with PUF for the scaffold project and biobrick TAL | ||
| + | |||
| + | *Entrepreneurship: Concern about the project progress. Send out our constructs for Patenting. | ||
| + | |||
| + | *Necessary PUF synthesis if PUF doesn’t get cloned by the end of July. Thinking about mathematics modeling. | ||
| + | |||
| + | *We will win the Best Hail Mary Award! | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet35" style="display:none"> | ||
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| + | Tuesday, 7/17/12 | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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.) | ||
| + | - 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. | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet36" style="display:none"> | ||
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| + | Thursday, 7/19/12 | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - Anthony - transformed and plated yesterday’s ligation, started another ligation in case those don’t work. | ||
| + | - 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. | ||
| + | - 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? | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet37" style="display:none"> | ||
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| + | Friday, 7/20/12 | ||
| + | - no advisor’s meeting | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - Angela - YFP (Venus) fluorescence! Also, it’s sequence has been confirmed. | ||
| + | - Divya - did PCR and will run gel later. | ||
| + | - Asha - PCRed more WT PUF for pBAD. | ||
| + | - Isiah - working on digestion of linear PSB1C3. Will later run a gel. | ||
| + | - Adi - miniprepped petduet and the K---- biobrick part (NCFP and CCFP). | ||
| + | - Cara - Waiting on the digest for more conclusive results | ||
| + | - Uros - tethering is delayed because a delay on one primer and no sequence of mutant PUF. | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet38" style="display:none"> | ||
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| + | Wednesday, 7/25/12 | ||
| + | - 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! | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - Ligation troubles - is it reagents or the materials? | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | |||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet39" style="display:none"> | ||
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| + | Monday, 7/30/12 | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - Cara - will be focusing on her final on Wednesday. Will resume work on Thursday. Might be considering sequencing. | ||
| + | - 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. | ||
| + | - Anthony - started new digestions for pBAD-wtPUF. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet40" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Monday, 8/6/12 | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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 =) | ||
| + | - 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! | ||
| + | - 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. | ||
| + | - Adi - is now updated with the PUF project, and now needs a PHAT project update. Needs them to work on the business plan. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet41" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Friday 8/10/12 | ||
| + | |||
| + | Angela: | ||
| + | - Successfully transformed two plasmids in the cell and this has been confirmed | ||
| + | - Have tested the expression of PUF in the cell | ||
| + | - The YFP that has been cloned functions as expected | ||
| + | - The artificial RBS does not work as well as the native RBS | ||
| + | - Has been in contact with Dr. Wong, will be on contact with him on Sun about mutant PUF | ||
| + | Anthony: | ||
| + | - Will be testing the fluorescence of all the combinations of the controls and testers | ||
| + | - Hoping to have the combinations tested by Sun/Mon. | ||
| + | Bob: | ||
| + | - Has been changing things on the wikio to reproduce what Calgary had on their wiki | ||
| + | - Change wiki background | ||
| + | - Working on making sure the changes to the wiki display the same in each browser | ||
| + | - 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. | ||
| + | Cara: | ||
| + | - Did PCR of TAL, will use PCR clean up then run on gel to determine size of the PCR fragment generated | ||
| + | - Performing overlap extension PCR to splice together pieces of BM3 gene that have the PstI site removed | ||
| + | - Making TB media to grow BM3 for characterization assays | ||
| + | Uros: | ||
| + | - Sent Brad a draft of PUFwt tethered to alphaGFP and he responded and said everything looked good and he changed the his tag | ||
| + | - Heard back from the representative for the first synthesis quote | ||
| + | - Emailed Courtney regarding the quote for the synthesis and the sequences to be ordered | ||
| + | - Waiting for two primers for RNA scaffold cloning to come in | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet42" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Friday, 8/24/12 | ||
| + | Asha - went to a conference on technical presentations, is creating the human practices video and powerpoints for outreach school visits | ||
| + | Isiah - helped out with scaffold, ran a PCR that didn’t work very well, will throw out and make more media | ||
| + | Anthony - been helping Angela with PUF constructs and running protein expression gels. Mutant PUF is working well and WT PUF not so well. | ||
| + | Bob - been working on restructuring the wiki alot, needs all the new/revised protocols, | ||
| + | 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 | ||
| + | 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 | ||
| + | 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. | ||
| + | Quad day - Divya and Asha working on a new poster and handouts, maybe wearing labcoats? (good or bad idea... debate) | ||
| + | 1-2: Isiah, Angela | ||
| + | 2-3: Anthony, Asha | ||
| + | 3-4: Bob and Cara | ||
| + | Jamboree prep - Clear your schedule for Sept 26 at noon because it’s the seminar presentation at IGB. | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet43" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Friday, 8/31/12 | ||
| + | Should we all come together on one project or continue in these multiple tracks? | ||
| + | - 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 | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - PHAT: trying to biobrick 2 of the enzymes. After biobricking them, will work on characterizing them. | ||
| + | - 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: | ||
| + | - control YFP/wt puf YFP (protet) with WT PUF (pBAD) | ||
| + | - control YFP/wt puf YFP(protet) with mutant PUF (pBAD) | ||
| + | -***control YFP/wt puf YFP (pBAD) with WT PUF (protet) | ||
| + | -***control YFP/ wt puf YFP (pBAD) with mutant PUF (protet) | ||
| + | -***control YFP/wt puf YFP (pBAD) with WT PUF (pet) | ||
| + | -***control YFP/ wt puf YFP (pBAD) with mutant PUF (pet) | ||
| + | - Lac Z (pBAD) with mutant PUF (pet) | ||
| + | - Lac Z (pBAD) with WT PUF (pet) | ||
| + | - 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. | ||
| + | |||
| + | Roles to fill: | ||
| + | - fluorescence testing: Angela | ||
| + | - biobricking everything: Isiah, with help from Adi | ||
| + | - RNA/protein gels: Uros | ||
| + | - cloning (not biobricks): | ||
| + | |||
| + | |||
| + | |||
| + | Advisors meeting | ||
| + | present: Kori, Brad, Todd, Ahmet | ||
| + | |||
| + | Upcoming deadlines: abstracts, safety questions, track selection due September 7th | ||
| + | - ongoing deadlines: poster, presentation, wiki (WIKI FREEZE is Oct. 3) | ||
| + | - regional jamboree: Oct. 12- 14 | ||
| + | |||
| + | Currently have: control YFP, wt puf binding site YFP, WT PUF. | ||
| + | - co-transformed control YFP + WT PUF, and then wt puf binding site YFP + WT PUF in DH5a | ||
| + | - 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). | ||
| + | - have we perhaps mixed up what we think is control and puf binding site YFP? | ||
| + | *** 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. | ||
| + | |||
| + | Will try in the future: | ||
| + | - subclone YFP into pBAD (was previously in protet) | ||
| + | - back up plan from UNC’s Dr. Wong: mutant PUF binding to Lac Z in BL21 | ||
| + | |||
| + | RNA Scaffold update: | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - 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. | ||
| + | - in the next week will try to purify mutant and WT PUF, and then do gel shift assays with them. | ||
| + | - need a distribution of work for all of this. | ||
| + | |||
| + | Writing the abstract (due Sept. 7) | ||
| + | - a paragraph, similar to what would be in a paper | ||
| + | - can write about things we haven’t performed yet, but need to be realistic | ||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | <div id="meet44" style="display:none"> | ||
| + | <textarea readonly rows="30" wrap="soft"> | ||
| + | Saturday, 9/15/12 | ||
| + | - 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) | ||
| + | - 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) | ||
| + | - tshirt design approved by all! | ||
| + | - powerpoint - next Friday will have a practice presentation for the advisors | ||
| + | - video - Bob has been working on animating through powerpoint. | ||
| + | - poster design - need to graphically represent all the data and constructs (takes a lot of time!), Cara has experience with this | ||
| + | |||
| + | </textarea> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
</div> | </div> | ||
Latest revision as of 19:24, 2 October 2012
