"
Page
Discussion
View source
History
teams
Log in
Team:UIUC-Illinois/Notebook/MeetingNotes
From 2012.igem.org
Revision as of 19:24, 2 October 2012 by
Bchen26
(
Talk
|
contribs
)
(
diff
)
← Older revision
| Latest revision (diff) | Newer revision → (diff)
Header
Home
2012
2012e
2011
2010
2010
Software
2009
2009
Software
2008
Team
Undergrads
Advisors
Blog
About Us
Overview
Project
Overview
PUF
RNA
Scaffold
Data
Results
PUF Data
RNA
Scaffold
Data
Biobricks
Ancillary
Overview
Petrobrick
Testing
Assembly
Line
Notebook
Protocols
Meeting
Notes
Lab
Notebook
Outreach
Overview
Safety
Overview
Attributions
Overview
Protocols
Meeting Notes
Pre-April
2/17/12
2/19/12
2/26/12
3/4/12
3/6/12
3/7/12
3/11/12
3/25/12
3/27/12
April
4/1/12
4/3/12
4/4/12
4/8/12
4/14/12
4/15/12
4/17/12
4/22/12
4/24/12
May-July
5/1/12
5/25/12
5/29/12
6/1/12
6/11/12
6/12/12
6/18/12
6/19/12
6/20/12
6/21/12
July-August
7/5/12
7/6/12
7/9/12
7/10/12
7/11/12
7/12/12
7/16/12
7/17/12
7/19/12
7/20/12
7/25/12
7/30/12
August-Jamboree
8/6/12
8/10/12
8/24/12
8/31/12
9/15/12
Select a date to read respective meeting notes.
iGEM advisors meeting 2/17/12 Meeting Present: Cara, Adi, Uros, Bob, Asha, Amanda, Will, Isaiah, Angela Advisors: Dr. Jim, Todd, Courtney Evans (Lab manager), Brad Evans (post-doc), Dr. Bhalerao Agenda: - Team formed in 2007, first competition in 2008 (four competitions) - Discussion: do we want to create a computational team? Involve open recruitment or connections. One of the teams at the finals had a computational team that created an evolutionary model of gene dynamics. (Prof. Bhalerao involved heavily in the computational side) o Bhalerao – it shouldn’t be done if it’s not 110% - The pep talk: Don’t lose commitment, you are chosen to represent Illinois. Don’t quit and do your job. o Bring it on! o Watch past presentations to realize the level of competition that we’re facing. They’re mind blowing. - 2 aspects to this experience: the synthetic biology (the idea, the research, the quality of the physical project), marketing (broad vision, best presentation, a sense of a dynamic vision is important, the judges like videos!) - Understanding synthetic biology: this is the big idea, understand it before anything else. Cut through the jargon. Take advantage of its flexibility. o When presenting to the judges, need to let them know that we’re doing synthetic biology, not nanobiology. - Meet with the advisors regularly to keep on track. Summary of Advisor’s labs: - Dr. Jin: Doing yeast and e. coli metabolic engineering. Engineer a pathway to create a phenotype of interest. All projects are about genetic manipulation and perturbation (also some environmental perturbation) in order to produce a phenotype faster, easier, cheaper. Will learn genetic manipulation, how to culture the cell in large scale and small scale (fermentation/bioreactors), mass spect to analyze the metabolic product. Also some computation in order to analyze results (but not a good idea to focus on in this time frame of 2-3 months). - Dr. Bhalerao: Pathogenic research. What happens to the stress response of the cell and the cell’s evolution in response to a metabolic engineering/gene circuit that you have put in the cell? Well it’s hard, so you look at evolution on the population scale. Can we control the evolution of the bug? If we so we can build better vaccines or harness these bugs for more work. (Example: can we control the evolution of pesticide resistant bugs?) Project: bacteriophage system. How does system change under different stresses? It’s complex to measure, so can we genetically modify these bacteria to visually tell us things? Will learn genetic manipulation, mathematical modeling, evolutionary genomics. - MMG (Brad Evans, Todd, Courtney): help contruct a series of vectors that will put tags onto the e.coli genome that will tease out their functions. How can we figure out what this protein does and how it does it? VERY iGem applicable. - Dr. Rao: similar to Dr. Jin, chemotaxis, as well.
iGEM Weekly Meeting – Sunday, February 19, 2012 Getting card access to IGB - Take 3 safety quizzes online (Angela will send out the link) - Take the certificate you get from this to the IGB gatehouse, first floor, and they will give you another certificate - Take this 2nd certificate to the iCard people and get your card access! - Do this asap! EOH - Will be going into the lab on next Saturday to keep up with the wet lab side of the e.chromi project (we are allowed to be in lab from Monday to Saturday) - 2nd project: coliroid. A red light system would need to be built so that we can illuminate the bacteria. (we already have the necessary e.coli strain). - Potential projects: making e.coli smell like bananas or wintergreen! Also, seeing strawberry/your own DNA. (these are all really good for kids) - The budget is $50. Making a nice printed poster is $80 through the IGB, so lets go simple (tri-fold, old posters, Adi’s computer presentation, hang up Asha’s dry erase board) Team Work Delegation - There are no longer any set positions. We are going to self-delegate tasks, but then we are also going to cross our own borders and help each other out. - Big idea! Everyone needs to work together, collaborate, and do their part. - We still do need a president, vice president, secretary, treasurer because we are an RSO. - The engineering council needs us to submit people for these positions. We’re going to choose these people, but we’re all going to collaborate. Isiah will send out the survey about this. - There is still the possibility to change positions if as time goes on we see that people have different strengths than originally thought. - Read the Pubmed “Leading a Successful iGEM Team” by Wayne Materi Presentations of Past Winning Teams - Adi’s article has been posted to the facebook page o E. coli were reengineered to be a chemical recording device. Once they were exposed to a certain stimuli, they would lengthen as a function of time. So the longer the e.coli were in length, the longer they had been exposed to the chemical stimuli. o Helpful in a biological context, also a nanorecorder for toxins or pollutants. o Possible project idea: rewire e.coli to detect any type of toxins for a specific environment. Another idea, not only does the e.coli change color when a toxin is detected, but it starts to break the toxin down. Research if past iGEM teams have done this. - First place: University of Washington o Used e.coli to produce alkanes, a major part of diesel fuel o Used a protease that digested gluten and made it able to digest more gluten (idea of taking pills with the protease in it so gluten intolerant people can take the pill) o Biobricks: magnetic e.coli, magnetism toolkit for future iGEM teams, submitted 65 biobricks! - Will talk about Imperial College London and JZU – China next week Project Ideas! - The above e.coli that detects and cleans up toxins - A harmless e.coli that goes in your body and detects trace amount of carcinogens or other toxins - Bioskins: gloves with e.coli on them that would change color letting you know what different types of chemicals are on your gloves or that there is too much dangerous chemical on your hands - Getting rid of carbon monoxide. Either in your home through a bacterial system, or putting it in factories to purify the exhaust air before it is released - Bacteria that make biofuels. Lets repurpose these to make them even more useful. Can we harness photosynthesis or put them in the smokestacks to purify the air? - Create a bacteria to keep invasive populations in check. (light sensitive so they undergo apoptosis during the day) - BUT – we need to research these ideas to make sure that we can actually execute these. - The iGEM categories: Best food or energy project, best environment project, best health or medicine project, best manufacturing project, best new application area, best foundational advance, best software tool, best information processing project, best human practices advance, best poster, best entrepreneurship - We REALLY want to register for both iGEM and the new entrepreneurial competition. Lab Assignments - Jin: Uros, Adi, Bob - Bhalerao: Isiah, Anthony - Brad: Cara - Rao: Divya, Asha
iGEM meeting minutes from 2/26/12 - We watched the first 13:00 min of this video about Imperial College of London http://www.youtube.com/watch?v=ushmgPM7HT8 o Please watch the rest of this video on your own- it’s very inspiring! o We are ahead of the schedule of ICL in their video; let’s not waste our time! - Angela suggests that we have two more meetings during the week in addition to Sunday for a total of 3 short meetings a week: o One to discuss team operation, one with advisers, one journal club meeting; each would be one hour in length o Multiple meetings would allow for more efficient use of team time and resources, we can solve problems from one meeting before the next meeting so we don’t have to wait a whole week to get answers - PLEASE FILL OUT THE “WHEN IS GOOD” SURVEY FROM UROS o this will select times for the two additional weekly meetings and give Courtney a time to come talk to us about safety - Safety Quizzes o Please complete your quizzes this week, we cannot work in the lab on EOH projects until everyone completes the quizzes o We are thinking of having a quiz party on Tuesday night this week to do all the online safety quizzes and have some food, check email/FB for a solid time o Please stop by the IGB Gatehouse 2nd floor Monday-Friday 8-5 (except the noon lunch hour) to get your IGB email address which is necessary for one of the safety quizzes - Journal Club o No article was discussed today o We are looking to get JC running during the week based on the results from the when is good survey- Anthony will lead first JC this week of 2/26 o Whoever will lead JC meeting of the week will create a small power point outlining the paper o Other iGemmers will read paper and come ready to listen and discuss - Adviser labs o Please email your adviser to introduce yourself so that we can get working in lab o Lab assignments: Dr. Jin Dr. Bhalerao Brad Dr. Rao Bob Isiah Cara Divya Uros Anthony Asha Adi - EOH o Equipment: projector, camera, computer IGB gatehouse, second floor, Melissa McKillip to obtain EOH presentation equipment o Presentation committee- Cara has the practice poster for EOH, we should discuss what other presentation materials we want to have at EOH o Lab: glowing bacteria (bright yellow/blue) as backup in case we all cannot get safety certified in time for EOH - Social outing suggestion: visit another team such as Northwestern, U Michigan, Wash U, Purdue to network and have fun - Discussed timeline for Spring and Summer o This semester learn lab techniques Journal Club- once a week to familiarize ourselves with the Syn Bio field so that we can create a stellar, cutting edge project o Summer 1st week: identify the final project 2-4 week: design the gene circuit - http://www.tinkercell.com/ will help you practice 5-12 week: lab troubleshooting o Overall- no specific time continue Journal Club Human Practices Wiki for the competition Have some FUN! - Announcements: o Publicity Director: open houses- once we do all of our work we would like to present it to as many ears as possible raise awareness of iGEM create flyers and brochures events that we need to prepare for: EOH, Undergrad Symposium, IGB fellow, Quad Day, Welcome events o Corporate Director: Talk with Melissa McKillip about funding sources: UIUC and industry If we get good funding, we can commercially synthesize some DNA sequences and save time! o Web Master: learn how to code iGEM Wiki now · Wiki is for the iGEM judges start Illinois iGEM website for meeting times, latest updates, and a place where the general public will come to look at our iGEM work- therefore it needs to be broad and understandable to the general public o Treasurer: needs to take RSO treasurer quiz - Uros presented the Imperial College of London websitehttp:// 2011.igem.org/Team:Imperial_College_London o take away points: liked the simplicity of their page they readily showed the implications of their project it was a global problem that had a wide area of impact they defined terms/provided links for definitions had lots of pictures and interactive features - Take a look at the Yale University project from 2011 that identified a very simple problem in a very simple way o this is an example of the beauty of simplicity o this Yale project was in the final for the regional jamboree and advanced to the international competition- don’t need to be intricate to win o we should take some hints from them!
iGEM Meeting Minutes 3/4/12 Announcements: Publicity - Divya - Everyone needs to sign up for an EOH shift on the google doc. Melissa and IGB have been contacted about equipment for a PowerPoint, posters, and 2 computers. An iGEM flyer is being made for EOH. - IGB fellows: a research symposium where 2 people will present a poster to IGB scientists. We will need people to cover shifts throughout the day. This is in a professional setting. Last year’s E. Chiver project will be presented. Social Chair - Cara - Everyone get excited for EOH! Social events will be planned after safety certification and EOH is done. A good idea is to do something before spring break. Corporate – Adi - Next week, a meeting with Courtney and Melissa will take place. Bob will accompany Adi to discuss getting funding for the website. Secretary - Asha - EOH posters will be worked on after this general meeting. Cara and Asha will be going to the first Engineering Council meeting this Wednesday evening. Lab Manager - Isiah - Lab work for EOH has been worked on by Angela. Last year’s project is still under consideration as to whether or not it will be included for EOH. - It is important for everyone to have access to IGB to come into the lab. Web Master - Bob - Bob has been looking at old wiki setups and has contacted David Chen from last year to help with this. - There will be the wiki page for project results and a different iGEM website for publicity and information. The judges only see the wiki. Another option is for the wiki to be hosted off of our own website. - Learning python would help with Tinker Cell. When we decide what we want to model, we can start looking at what we specifically need to learn of python. Treasurer - Anthony - Needs to receive the account number from the last year’s treasurer. Vice President – Uros - Everyone needs to complete the EOH training and print the safety inspection sheet in order to be ready for the Friday morning safety check. - Everyone also needs to meet with Courtney to complete the IGB safety training. President - Angela - Next advisor’s meeting is Tuesday, March 6, 3-4 PM at IGB. We will be discussing why last year’s team was not fully successful. - Next Sunday, Angela will go over the E. Chiver project from last year. And every Sunday someone will go over another project. - Next week’s article: “A sensing array of radically coupled genetic ‘biopixels’.” It will be presented by Uros. Discussion: 1) Safety quizzes and IGB keys a. Keys are not working after hours or during the weekend. This problem has been brought up to Courtney. b. This Tuesday Adi, Bob, Anthony, and Asha will meet with Courtney to complete their safety training. c. So far only Cara, Uros, and Isiah have an IGB key. d. Procedure: d.i. Take all three safety quizzes d.ii. Go to Courtney for 30min IGB specific safety presentation d.iii. Get the access paper signed by Courtney (can ask her for a copy) d.iv. Go to 1st floor of Gatehouse to the front desk, tell them you are iGEM and need access d.v. Pay the $8.50 and get a receipt d.vi. Take your receipt to the Union Bookstore on Wright, go to id center in back d.vii. Tell them you need access to IGB and give them the receipt from the Gatehouse (be prepared to take a new id picture!) d.viii. Finally make sure you go to your residence hall front desk and get your new icard re-activated d.ix. Make sure your new icard will give you after-hours access, maybe go to IGB front doors after 5pm and see if you can swipe into the front doors 2) New meeting Schedule a. General meeting: We will still meet every Sunday from 3-5. For the rest of the semester, the Chemistry Library will be our meeting place. b. Journal club: The journal club will meet every week after the general meeting. We have 5 papers that the advisors suggested we read. c. Advisor’s meeting: First advisors meeting will be this Tuesday, March 6th from 3-4 at the IGB. 3) Has everyone contacted their lab advisor? a. If not, do that asap! 4) EOH progress a. We will meet after this to familiarize ourselves with the presentation for EOH. b. Everyone needs to partner up with someone who knows the presentation, that way at EOH we are prepared. 5) Journal Club (Presented by Anthony) a. “Synthetic biology: advancing biological frontiers by building biological systems” b. PowerPoint is attached to the email of these minutes.
Agenda of Advisor Meeting 3/6/2012 Team Structure - President – Angela Chen - Vice President – Uros Kuzmanovic - Secretary – Asha Kirchhoff - Treasurer – Anthony Chau - Corporate Director – Adi Malik - Publicity Director – Divya Tankasala - Social Chair – Cara Schornak - Web master – Bob Chen - Lab Manager – Isiah Ramos - But it is everybody’s responsibility to contribute and direct themselves, as well as collaborate with all the others to make the project a success Team Operation - Meetings o Bi-weekly advisor’s meeting Tuesday from 3-4 PM o Weekly general meeting Keeping track on team operations Discussion on one past project o Weekly Journal club - Recent Activities o Participated in E-week events o Preparing for Engineering Open House In order to use this in the human practices part of our project, we will video tape people’s opinions on synthetic biology, video tape the exhibit, and count the number of people we interacted with A short edited video can then be used for the human practices part of our website Overall, the responsibilities of human practices falls to Divya, Cara, and Asha o Completed safety quizzes Make sure Courtney has a copy of your completed safety quizzes! This is absolutely necessary to do before you get your IGB card access. o Started Journal club on March 4 Important to critically examine the articles, not just read them. We have to sharpen our critical thinking. We also should read review articles for every paper. o Started project discussion – 2011 U of Washington and Imperial College London o Started Bootcamp in each lab Every week everyone should share what they are learning in bootcamp. This will lead to brainstorming. o Presented E. Chiver project in the 2012 Institute of Biological Engineering on March 2 Angela and Jaimie recreated the old presentation so that it was more suitable to a symposium. The presentation was filmed, so it will be sent to all of the advisors. Discussion - Bi-weekly meeting with advisors o What do we discuss in this meeting? Present our ideas for projects to the advisors so they can give us an indication of how we are progressing and if our plans are viable. o Introduction to Prof. Liu of Bioengineering as one of our advisors. - Team Operation o Would identifying a topic in the first week of summer be too late? o Brainstorming Rules: · No idea is bad. Especially for the first 2 weeks, there should be no filter. Everyone should have the chance to speak. · After a few weeks start editing the ideas. Check iGEM.org to make sure that no one has done our idea before. This is also another good resource to check and see what type of projects went well and why. o Dr. Bhalerao advises to skip looking at the past projects of UIUC iGEM or other iGEM teams. Courtney agrees that we will never be proficient in synthetic biology, so it’s important to just come up with good ideas (which is the hard part). Looking at past presentations and projects is what we should all do individually, not at the meeting. o Our project should be feasible, something we enjoy, and something really cool. Our presentation should also be able to market and capitalize our idea, as well as convince everyone that it is better and more exciting than anything else. o Look at our old past projects and see if we can finish any or do any better. - Team Collaboration o Is collaborating with the University of Michigan team, or others, feasible? Try to build bridges with as many teams as possible. That way we have as many options as possible. These contacts need to be made soon. Our collaboration can be pooling human practices, sharing data or technology, or even sharing a project. It is possible to actually collaborate on the same project, especially if one lab blind tests the other’s idea, or one team plans and the other executes. - iGEM registration o Registration is now open. Do we have an iGEM account? Yes. o Can we join iGEM – Entrepreneurship? A good idea, ultimately we do want our project to be applicable in real life. We need to acquire an additional advisor from the business school. Try Kathy Baylis in Agriculture/Consumer economics. She is a friend of Dr. Bhalerao’s and her husband is a professor in the business school. Dr. Bhalerao thinks we should pursue the economic models track. This has the same registration deadline as the normal iGEM competition, which is March 31. - Funding Sources o Where do we find funding sources? We start from scratch every year. The only one that is guaranteed is IGB. We should start early. Funding went well last year; we’re starting with $15,000. But we should knock on all doors. o Should the corporate director and president talk with the IGB director? Yes, make a formal appointment and have an impressive presentation. Dress down for it and make them give you money. Also, make a brochure for iGEM 2012 and then go and talk to all the heads of all the Colleges. We can print it on nice glossy paper for free at IGB. Our brochure should contain as specific as a project idea as possible. We want to let everyone know that we plan to deliver. Also market that we want to join the Entrepreneurship competition this year. Also, contact Peter Fox. He owns the land that is Research Park. Also contact Scott Pilkins, the director of Research Park. Illinois Ventures in particular is one we should talk to. Steven Slyger from Nanodisk at Research Park. o In May, we will all need to fill out a form for travel reimbursements ($700). - Server o Do we have a server for the iGEM website? We do, Dr. Bhalerao has it in his office. We would be able to secure $100 for a high quality website from Adi’s Chicago web developer contact. But we should make sure it has dynamic contact. It it’s just static, we shouldn’t spend the money, and we should just use a template. - 2011 iGEM project o Why didn’t we advance to the international competition? There was no data uploaded. It was a human error. A lot of people liked the idea, but there wasn’t enough data and characterization to show the judges. The idea was too complex, the execution was poor. The project never really came together, it had no final polish. This year the goal is to ADVANCE. o How should we manage time/human resources to complete a winning project? Towards the end of the project, everyone needs to continually be checking and double checking that everything is correct and has been submitted. We really don’t want to let our wiki go until the last minute. Ideally it should be created over a long amount of time. We also really want to focus on our presentation. We should memorize it, but then make it seem effortless. Things that stand out: great idea, great execution, and obvious team chemistry.
3/7/12 Engineering council Present: Asha, Cara Officer announcements - Engineering open house: o Thursday night – you must attend check in at Kenney Gym starting at 6 PM! T-shirt pickup, equipment, and signs will be given out at this meeting. The final safety meeting is at 9PM after this. o We need to volunteer 4 hours as a society, and register on the EOH website (attractions tab, volunteer, google survey and doodle). o Fun stuff: Tesla Coil Concert at 9 PM on Friday, Dorm room fire simulation (11, 1, 3 PM on Friday only!), James bond exhibit. Friday night speaker is Danny Forester (host of Build it Bigger on Discovery), but this needs tickets which you get can on the hour every hour at Grainger. Also, you can download the EOH app. - EC executive board elections: society should decide on who to vote for once the names and statements are sent out. - EC/BC barcrawl (engineering and business council). Wed, March 14th at 9 PM. Can sign up on facebook - March madness bracket challenger: $5 per entry with money going to Habitat for Humanity. - Committed Student Award: We can nominate one student for significant contributions to an EC society. Application available online. - Town Hall meeting with Dean Tucker (dean of undergrad engineering) is Monday Match 12th, 1320 DCL at 5 pm. Free food! - Undergrad Research Workshop: March 13, 11-12 in 112 Engineering Hall. - How to make your own nonprofit: March 14, 5:30-7 PM in 269 Everitt Hall. - Rock your internship: March 27, and grad school workshop after the break - Awards: o Committee member of the month: Sid Sethupathi (Eweek mastermind and a freshman) o Society Member of the month: Joseph Shim of IEEE o Society of the Month: AlChE (E week champs) o Yearly Awards: Most improved society: IIE o Yearly Awards: Outstanding Society: SWE
iGEM Meeting Minutes 3/11/12 Announcements: Publicity - Divya - Film and pictures from EOH will be posted to the facebook page during spring break. The brochure template will also be created over spring break. - Melissa wants iGEM to contribute a little blurb to the online newsletters. Asha will be writing that. Social Chair - Cara - Is planning a game night before spring break. Details will be posted on the facebook page. Corporate – Adi - Not present, look for an announcement through a separate email. Secretary - Asha - Still need to send minutes for the ec. Meeting, and last advisor’s meeting. Lab Manager - Isiah - After spring break, will be in charge of cleaning the lab bench and desk. However, everyone will help with both of those. - Also after spring break, Isiah and Anthony will collaborate on ordering supplies, and then it must be checked with Angela for approval before actually ordering things. Web Master - Bob - Will be moving to Bhalerao’s lab. Treasurer - Anthony - Anthony and Isiah have started lab work with Bhalerao. Vice President – Uros - Is presenting the journal club today. President - Angela - Good job on EOH everyone! =) Discussion: 1) What did you learn in lab bootcamp this week? a. Anthony and Isiah (Bhalerao): has a general overview of the lab with oral instructions. Biohazards were discussed and a gel was created. Actual lab work will start this week (at least twice a week). They will write a proposal for a procedure on how to extract, characterize, and transform a specific gene. Bob will also be switching into Bhalerao’s lab. b. Asha and Divya (Rao): No contact yet, we will definitely try to meet this week before spring break. c. Cara (Brad): Lots of stuff! Working hard over the past few weeks, Cara has been performing electrophoresis, ligation, and transformation. 2) Meetings schedule a. Over spring break, everyone should review past projects and come up with their own proposal for a project. a.i. Group 1: Angela, Asha, Bob, Isiah , Anthony a.ii. Group 2: Divya, Uros, Cara, Adi a.iii. There will be no discussion on the past projects, but it is important for inspiring our own project proposals. b. Next general meeting is on March 25 from 6-9 PM. It’s long because we will be discussing proposals and projects. However, it will be in a social setting with food! b.i. There will be no journal club! b.ii. Group 1 will take 20 minutes to present their proposal. c. Next advisor’s meeting is on March 27 from 3-4 PM in IGB. c.i. Group 1 will present again, and the advisors will give their critiques. d. Weekly meeting on April 1 d.i. No journal club! d.ii. Group 2 presents their proposals e. Advisor’s meeting on April 3 from 3-4 PM at the IGB. e.i. Group 2 will present their proposals again. 3) E. Chiver was presented at the IBE conference. We watched the presentation that Angela and Jaimie gave. (Attached to the email of these minutes). a. It was a good novel idea, but it was not possible to transform that many genes with the small number of people who had lab experience. b. So for this year, we want to characterize one gene really well and then build up from that simple concept. b.i. Another good idea: find another concept and make it easier or better in some way. 4) Journal club: “A sensing array of radically coupled genetic ‘biopixels’.” Presented by Uros a. Presentation attached Email list: Adi Malik <adi.malik24@gmail.com>, Angela Chen <chen398@illinois.edu>, Anthony Chau <chau3@illinois.edu>, Asha Kirchhoff <kirchho2@illinois.edu>, Bob Chen <riboflavinbob@gmail.com>, Divya Tankasala <divzmyster30@gmail.com>, Isiah Ramos <greenstatic92@gmail.com>, Uros Kuzmanovic <ukuzma2@illinois.edu>, manda68@gmail.com, jones28@illinois.edu, ysjin@illinois.edu, chrisvrao@gmail.com, bhalerao@illinois.edu, courtfuentesevans@gmail.com, brad_ev02@yahoo.com, freesto1@illinois.edu, joannema7@gmail.com, mmckilli@illinois.edu, luting@illinois.edu
iGEM Meeting Minutes 3/25/12 Announcements: - Thanks for the wonderful food everyone! Special thanks to Cara for organizing this and Anthony for hosting! - Presentations today! - Only 6 weeks left! There will be no meetings in May so everyone can focus on their finals, so we have to make good use of the 5 general meetings we have left! - Things to do before summer: o Clean up the lab! Angela started, but we need to dispose of all the old chemicals and strains. We also need to make an inventory that will be digitalized. Isiah will be in charge of the lab, but we are all going to help! Things to be cleaned: 4 degree room for fridge, -80 and -20 degree rooms for freezers, lab bench, and lab desk. o Familiarize ourselves with the protocol that Angela will send out. o Familiarize ourselves with the lab machinery. PCR, gels, centrifuge. Get to know these in lab bootcamp. o Familiarize ourselves with biobricks. We’re going to try to create a circuit based on previous year’s biobrick kits. Also, the biobricks are free so if you want one, just go to the part registry to order it. The email for ordering is hq@igem.org. o We want to find sponsors. Divya and Adi will needs to collaborate to create a brochure and start contacting corporate sponsorships. This will really come together after we figure out what our project is. - Upcoming publicity events: o Undergrad research symposium: Divya will be contacting us about when this it gets closer. o Volunteering to do chemistry demos with Courtney sometime in mid to late April. Send Courtney an email if you are interested! - Collaboration with other teams: o We have been contacted by University of Michigan to collaborate. We will see how this progresses as we both come to conclusions about our separate projects. o During the summer our goal is to go visit WashU, Northwestern or both! We will take a weekend off during the summer to have fun with another iGEM team! Anthony and Cara will collaborate on this contacting the teams and getting SORF funding. - General outline of summer schedule: o There will be a short daily meeting that is mandatory! This is just so we all know what everyone else is doing and can keep on schedule and organized. o We will have deadlines, but flexible lab hours. Come in Mon-Fri as your work requires. The goal is to lead ourselves and be self-sufficient. o Our first day in lab is May 16. We get a short little break after finals to recharge and get ready for a great summer in lab! o May 19-20, the first weekend we are at the IGB, we will have meetings on Saturday and Sunday! This is the only weekend we will have meetings. o We will also have weekly advisor’s meetings. Courtney will not be present, but will communicate with us through email. o During the summer, we have 7 days of travel breaks. You MUST let Angela know if you want to take more than a week off. This seems like a lot of work with no breaks, but we want to work hard to try to complete a great project! If we finish early, there will be more free time towards the end of August. o We will finish the wiki during summer! We will also try to completely finish the project during the summer. o Human practices will be completed during the summer under the leadership of Asha with assistance by Cara and Divya. However, everyone should help! o We will have social events during the summer. We want to bond and manage our stress! Presentations: **** Note! Our next advisor’s meeting is Tuesday from 3-4. All presentations will have 5 minutes to talk. A good idea is to start with an elevator pitch. This will be followed with 5 minutes of discussion. This year the plan is also to choose multiple projects. So we need ideas – no idea is bad! If you would like to present 2 ideas, you will be able to present at both advisor’s meetings. **** Anthony – Production of a New Food Source for Aquaculture of Predatory Fish Isiah – Bacteria Refined Oil Adi – Biodegradation of plastic by reengineered E. Coli Asha – Tuberculosis fighting E. Coli Bob – Electrical energy from photosynthesis Uros – gives everyone useful articles/links to past iGEM projects Angela – Engineering RNA site specific binding proteins
iGEM advisor’s meeting 3/27/12 Project proposals: Angela: RNA Site- Specific Recognition Machinery Using PUF - It is know that PUF bind the RNA and assists with gene expression. However, not many other details are known. - It’s like zinc fingers for RNA - Is 8 base pairs specific enough to identify viruses? What if we used several PUFs? 16 base pairs would probably be enough. This works well as a viral detection system. - RNA detection system is a possibility but it would take a long time to perfect. - Use possibility is an in vitro RNA regulator. Binding of PUF to mRNA inhibits its expression. Very simple, and a proof of concept experiment. This could tie into the lysin manufacture for the tuberculosis proposal - Want a quick easy test to see if this works. This is definitely worth pursuing. Anthony: Production of a New Food Source for Aquaculture of Predatory Fish - E. coli naturally make triglycerides, but not the type we’re looking for. There is the possibility to introduce another substrate. - Is there the possibility to create a bioreactor where the enzyme just breaks down fats? - More research on possible substrates needs to be done. Asha: Tuberculosis fighting E. Coli - Wouldn’t work with tuberculosis, would use M. smegmatis which has 17 known phages - It takes 2 weeks to grow! It’s hard - Perdue might be pursuing something like this because one of their professors has done research on it. - Instead of looking at tuberculosis phages, just look at bacteriophage ideas in general. - Need to look at the lysin proteins in more detail. Bob: Crossing the Self-sustainability of Photosynthetic systems with Other Cells - It’s not really possible to introduce photosynthesis to e. coli. - Nanotechnology or endosymbiosis would be better options - We need a model for this Recommendations: - Do the PUF project and then pick another one of the 3 application projects to pursue. Announcements: Divya – publicity: - We are signed up for IGB Fellows, where the E. Chiver poster will be presented. 1 or 2 people need to present, so watch for the email closer to May. Cara - WashU has been contacted, Cara’s contact is working to connect us to the WashU team. - NU will be contacted next, along with Perdue Angela - This Saturday we will be cleaning up the lab! Everyone should come to help and learn more about where everything is in the lab. It will start at 1. - The coming weekend meetings are very important, please commit the time to be there! - On April 10th we will meet with Dr. Robinson, the head of the IGB. You have received an email with the details of this. o Aside from this meeting, every Tuesday until May will be an advisor’s meeting.
iGEM Weekly meeting 4/1/12 ***Important! Please be punctual! With so few weeks left, every minute counts! *** Announcements: Adi, Corporate: - Contacting academic departments of bioengineering and ag&bio engineering to see if we can obtain funding. Dr. Amos (undergrad BioE head) has been contacted. She said that she might not be able to write us a check, but the BioE facilities might be opened to iGEM. A meeting is scheduled for this/next week for Angela, Adi, and Dr. Amos to meet. - by the end of summer, companies will be contacted as well. (Fischer Scientific) - Website: Alex will have a template created by the end of April. Adi has sent him Harvard, Calgary, MIT, and Imperial College London as examples. (Calgary and Imperial are both best wiki winners). Adi is putting in a request so that this will be done in PHP, that way our wiki can be dynamic. - Only 5 other teams have signed up for the America division of the Entrepreneurship competition. This is very promising for us! Bob, Web master: - Has met with David, the web master from last year. Bob’s going to get to experiment with the template from last year on another server. The link to this has been put on the iGEM facebook page. Divya, Publicity: - Undergraduate Research Symposium is on April 11th! Volunteers are much needed, so email Divya if you can make it for just a few hours. - A workshops on how to effectively present posters are also going on this Wednesday and Thursday at the UGL. Come when you can and let Divya know about it! (This event has been added to the iGEM google calendar.) - iGEM is going to get registered for Quad day next fall. Isiah, Lab manager: - Need to start deposing old materials in lab and cleaning the bench. That will happen this week. In the following weeks we will streak stuff out to see what is functional. The 4th week, everyone will send their results to Isiah who will compile the data into 2 different books that will allow us to be effective during summer. - Times for this week: Monday 11 AM – 5 PM, Wednesday 11 AM – 4 PM if necessary, Friday after 11 AM. We are NOT allowed to be in the IGB after 5 pm. Watch for Isiah’s email that will follow up to this. Cara, Social: - No news back from WashU, and there is currently no IGB money to travel to any other colleges at all. - Cara and Asha will look into traveling funds from Engineering Council. Anthony, Treasurer: - If we get SORF funds we will get reimbursed for all of our travel expenses. So we will have to pay for everything upfront, but once we present a receipt we will be endorsed. This will not cut into our IGB funding. - This will be discussed with Courtney in the next advisor’s meeting. Uros, Vice president: - Uros had the same idea as Adi. Dr. Jin suggested creating magnetic organisms – yeast that are magnetic. However there are not many applications. Uros will be further communicating with Dr. Jin about this and brainstorming ideas to come up with a proposal. In the meantime, Uros and Adi will be collaborating on the project proposal. - Since degrading plastic is a popular idea, we will investigate to see if any other teams are going to be pursuing it. If so, then we will try to collaborate with that university’s team. Asha, Secretary: - Please email me your powerpoint presentations for your proposals! Also, post them to poptab. - Cara and Asha will be attending another Engineering Council meeting this week. We will be voting for Engineering Council positions. You have all received an email with information on the candidates, so please send your opinions and feedback to both Cara and Asha Angela, President: - Everyone needs to go officially sign up for the UIUC-Illinois iGEM team. Courntey has sent everyone an email with directions on how to do this. - In April, we will be working on finalizing our idea. So work on your proposal further! Review more literature and refine the proposal in order to make it even better. - If you have yet to present, send Angela a title and a summary of your proposal. - Overview of the Parts Registry: o Where all the biobricks are o At the beginning of the summer we will get a kit with about 500 biobricks that we can use in our parts. o The parts registry shows the parts by type with information about each type of part. Green W’s means the part works! o Upper right hand “get this part” is where you can order the part. Be sure to note the part name, the plasmid backbone, and what kind of antibiotic resistance it has. Ordering any part is free! o Computer modeling: perform a scratch where we put the sequences together to check them before we got to the lab o Before summer, spend some quality time learning what all the different types of parts are and how to put them together. But don’t memorize everything – it’s just not possible! Also review iGEM standard cloning protocol. Angela will take an hour next meeting to give an introduction to cloning and standard protocol. - Next week’s meeting: Will be sometime Sunday NIGHT due to Easter. Check the facebook group for more details. There will be no journal club. Still bring ideas and develop our existing ones! Develop further proposals. Proposal presentations: - Divya – Transplastomic antibacterials o Plant derived antimicrobials: traditional antibiotics, MDR inhibitors, Compounds that target bacterial virulence o Genetically modified plants have new genes inserted into the chloroplast DNA. o We create transgenic plants that make drugs. o We need to find someone on campus with a gene gun. - Cara – A Privy Understanding of E. Coli o After natural disasters or in underdeveloped areas sanitation and uncontained sewage is often an issue. We need a quick, inexpensive, simple solution. o Create packages of freeze dried bacteria that will be able to handle high volumes of human sewage. But the bac in a bucket with water or direct sewage, and a color indicator shows when the sewage had been purified and it is safe to dispose of the remainer of the waste. Color goes from blue -> colorless. o The kill mechanism is sunlight. When you throw the newly processed waste water out, the bacteria die in the sun. o Good project for human practices and for entrepreneurship; need to target/focus the pathway more.
iGEM Advisor’s meeting 4/3/12 Proposal presentations: Uros and Adi: “Biodegradation of Polyester Polyurethane by Endophytic Fungi” - Isolate the genes that degrade plastic and put them together in E. coli to breakdown plastic at a commercially viable rate - Alternatively, certain strains of fungi have the ability to degrade polyurethane plastic. - We would focus on one plastic composite to degrade. That would make the project more feasible for one summer. - E. coli might not be able to handle all of this. It’s been proven that the process works in anaerobic and aerobic conditions, so yeast might be another object. - We have the capability to build the type of filtration system necessary. - Is it the best to degrade this plastic? It’s carbon sequestering. The degradation of plastic would need to be coupled to something that would get rid of the CO2. We could couple it to make a precursor for some new plastic. We would need to repolymerize it in a type of biological recycling. - Plastic is good to recycle. Another way to think about this project would be to microbially recycle plastic. Divya: “Transplastomic antibacterials” - Plants make small molecule compounds that act as antimicrobials against Gram positive bacteria. There compounds typically act in combination. - Genetically modified plants would produce antimicrobials. New genes are inserted into the chloroplast DNA. - Use a gene gun to deliver the DNA to the cells. This way the chloroplast’s double membrane is penetrated. - Rate of transformation of plants is very low and growing plant tissue is difficult and takes a long time. - Transforming plants is a task in and of itself. We do have resources on campus to do this. It would look very impressive if it actually worked. - Great project, but we need some feedback from alternative sources about the gene gun and plant transformation. - This is a complex project for the time frame. You have to isolate the compound, look at the genes, then isolate and transform. This might work as a side project though, if a very simple compound was used and we changed its expression levels. Cara: “A Privy Understanding of E. Coli” - Fecal matter is the main source of bacterial pathogens in waste. Create a little package of genetically modified bacteria that would be added to a bucket that would be used as a toilet. Bacteria would sanitize the fecal matter and a color change would indicate full breakdown. - How to focus the project: Choose one compound to degrade, look at what types of bacteria/fungi/protozoans are in the mix. - Animal feces might be a bigger problem than human waste. - The bacteria sanitizing the fecal matter would have to compete against the pathogenic bacteria already in the environment and fecal matter. - Look up basillophila. Announcements: - In the next 2 advisor’s meetings, decide which projects are the best ideas, focus on those, and represent them in groups. Have different people both critique and endorse the different projects. o Rank the projects. - There will be one main project, and then one good side project. o The side project functions as both a safety in case the primary project fails, and as a continued project for the next year. - Keep in mind what looks good in competition. The work we’ve put in might be enough, even if there is no finished final project. But the research and groundwork must be excellent in that scenario. - Final advisor’s meeting is Tuesday, May 1. At this meeting the main and side projects will be finalized. - Next Tuesday we are meeting Dr. Robinson. - April 17th, we will have a group picture for the brochure. Dress up! - On Sunday’s meeting we will vote on what projects we will focus on pitching again. This is the opportune time to add more to your presentation. We’re not picking 2 projects, we’re picking several to re-research and reevaluate. Remember, we’re objective and no project is a bad project! - This Sunday Angela will also explain about the standard cloning procedure. - Sunday meeting will be at 8PM in the BIF.
iGEM Engineering Council Elections 4/4/12 - We removed 10 inactive groups (they had not communicated with EC for a full year) - Amendment passed, says candidates on co-op or study abroad can make a video communication to run even if not physically present at the EC meeting. - Gamma Epsilon general engineering honors society appeals to come off of suspension o There is drama and arguments from both sides o Written, secret vote They are still on suspension. - Election results: o Pres: Courtney O’Connor o VP: Patrick Kennedy o EXPO: Elena o EOH Director: Gloria Lin (voted to allow a non-engineering on EC) o Director of Leadership: Steven Marks o Secretary/Treasurer: Troy Meeham o Dean’s Student Advisory’s Committee Director: Akash Shah o Future enrichment opportunities: Kritika Jetkey o EIB Director: Douglas Podgorny o SITE director: Mary Kate Krouse o Awards director: Samantha Tone o Publicity Director: Rachel Gross o Social Director: Rachel Seidner o Service Director: Sara Moshaga o Director of Information: Sid S. o Knights of Saint Patrick: Christopher Massie
iGEM weekly meeting 4/8/12 Announcements: Isiah, Lab Manager – The next 2 weeks is about organizing the lab. We need to divvy up the remaining work. Expect Isiah’s email about the inventory in a few days. Isiah will just finish cleaning a few more things up and then the lab will be ready to go. 2 books will be created, one for the glycerol stock and another for primers/buffers/ etc. There will be a physical copy in lab and one digital one. Cara, Social, & Anthony, Treasurer – Thursday at 4 PM is the deadline for applying for SORF. WashU has not responded to Cara, so we will try to use Uros’ biobrick contact. We will not be able to take more than 2 trips, and 1 trip would be awesome and enough work. This trip would be great for human practices and collaboration. Cara will contact Northwestern. U Mich will ultimately collaborate with us, so that’s our option. We will apply for the money anyways, because even if we don’t use it the money will just get reallocated back to the SORF fund. The application will be about how the trip helps our human practices collaboration. Cara & Asha – contact Courtney O ‘Connor about funding deadlines. EC funding works similarly to SORF, but we need to check deadlines about that. Asha will be emailing Courtney (outgoing) and Troy (incoming). Results of the EC elections: o Pres: Courtney O’Connor o VP: Patrick Kennedy o EXPO: Elena o EOH Director: Gloria Lin (voted to allow a non-engineering on EC) o Director of Leadership: Steven Marks o Secretary/Treasurer: Troy Meeham o Dean’s Student Advisory’s Committee Director: Akash Shah o Future enrichment opportunities: Kritika Jetkey o EIB Director: Douglas Podgorny o SITE director: Mary Kate Krouse o Awards director: Samantha Tone o Publicity Director: Rachel Gross o Social Director: Rachel Seidner o Service Director: Sara Moshaga o Director of Information: Sid S. o Knights of Saint Patrick: Christopher Massie Adi – Meeting with Dr. Amos Monday at 8 am. We’re not looking at funding, but using facilities instead. Website new, Alex has been sent designs from past years and he is now requesting filler data and specifics on what we want included on the website. Everyone should write a short bio and find a picture to send to Asha. Asha will compile these and the meeting minutes to send this to Adi and Alex. Send these by Friday. We actually did have a website in 2010, so that will be consulted. For entrepreneurship, we need to find a faculty advisor. Bob emailed Rao’s contact, but she never followed up. Angela will contact her professor who is interested in bioethics. We also need to register for the entrepreneurship competition in the same way that we applied to the normal iGEM competition. Apply without the membership code and Courtney will approve of us later. Divya – practice time for undergrad research symposium will be on Tuesday. Uros, Angela, and Divya will be presenting. The symposium is on April 11, from 9-3/4. It is an hour poster presentation. We are now registered for quad day. Bob – Has created an entirely new page to start the website from scratch. Angela – For the next advisor’s meeting we are meeting Dr. Gene Robison in the first floor of the gatehouse. From 3-5. Dress casually, but not sloppy. At the end of the meeting if time permits, Courtney will discuss how to keep a standard lab notebook for us. April 28th is the chemistry outreach demos that Courtney is doing. The demos will be for high schoolers. Example demos are the vomiting pumpkin (elephants toothpaste) and the exploding gummibear. We will be going to Rantoul for this, but transportation is providing. Ideas for demos: color changing solutions, liquid nitrogen/soap/water, make a pickle battery, fire in a pumpkin. Cara will contact Dr. Ray of Chem 104 for demo ideas and protocols. Concrete times and a date for practice will be announced at the next advisor’s meeting. Entrepreneurship and human practices needs to draft a proposal. These proposals will get presented to the advisors on April 24th. On April 22nd we will practice these presentations. Agenda: UIUC-Illinois Judging Form - Bronze: submit a biobrick - Silver: characterize biobrick - Gold: Characterize a biobrick that is not for our team. - Be sure to familiarize yourself with this! We’re going to continually consult this during the summer. We are going to get a gold medal! Lab updates - Bhalerao: Isiah and Anthony prepared electrocompetent cells. The experiment will begin Monday/Tuesday this week. - Jin: Last Wednesday Dr. Jin assigned us to post-doc Dr. Lee. Tomorrow Uros will meet with Dr. Lee to plan how the project will be executed. Tuesday from 9-11 everyone will meet to replicate the experiment. - Brad: Cara has been working hard every day. She transformed some cells with 3 different plasmids that each have a his tag and then either GFP, FoA, or LacZ. The LacZ failed and is being redone. - Rao: Angela is working with 2 operons to see if a deletion will effect expression. She tried out 4 different cloning methods. If the PUF project is done, one of 2 specific cloning methods needs to be used. Proposal discussion: - Next Tuesday is the second proposal round. - Isiah: Original idea: Use genetically modified E. coli to modify crude oil/biofuel to refine/recycle oil. Naturally occurring bacteria would be used. Further research indicates that time issues make this project infeasible over the summer. Current research being done has revealed that it is really difficult to grow this stuff in the lab. Also, one of the reagents to get the bacteria to grow is very harmful for humans. o Will’s feedback: For anything biodiesel look at University of Washington. This is a complex proposal, so to make this successful go look at past successful biodiesel projects and try to improve it/continue it slightly. - Uros: Magnetic bacteria. o Will: Can we use magnetic bacteria in a machine? Use b fields to control the bac or use the bac to signal things through b fields. These are ideas of applications. - Adi: Degradation of plastic composites. o Will: An Australian team has worked on this before. o Potential for a great human practices project: Write reviews on different topics (biofuels, sensing, etc) to help future iGEM teams orient themselves each year. That way it makes it easier to see what work has been done before. Also, we could devise a strategy to organize and exercise quality control over the parts registry. Start with a call/teleconference survey and then recompile. o Stanford-Brown created a human practices website and an alumni website. Max Song did this, and we’re going to try to contact him for further information and collaboration. - Angela: update: she has all of the PUF protein sequences and the RNA/aa info. It can be synthesized by a company (time and money) or self-synthesize (2 weeks, little money). University of North Carolina is researching the PUF protein and so we could ask for a template DNA PUM1 gene from them. Jin’s lab could also PCR out the PUM1 gene that expresses the PUF protein. So overall there are 4 different sources to get the gene. Translational repressors are an application. GFP can be used to characterize. Small side project – design and optimize a construct for the project (to be presented in the next advisor’s meeting). - Divya: looking at things with microbial properties, but not plants. Maybe yeast. - Bob: Found a part that uses light to run an ATP pump. We could look into photosynthetic bacteria rather than interfacing the electro-animal/slug with bacteria. - Uros: There are fungi/bacteria that can degrade plastic via serine hyrdolase. Yale is probably going to do this project for iGEM. It’s a simple but pretty cool project. We can anticipate that this will be a popular idea. Collaboration with Yale is an option. - Anthony: Looked into the price of the substrate ARA, but is unsure about how much is needed. Also, there are troubles getting e. coli to take up the fatty acids. - Asha o Broad review of how tuberculosis is treated. Look for someone on campus who is researching on how to treat tuberculosis/bacteria. Look for a direction to research further. - Cara: o An Australian team has put e. coli in sewage water before. It would be a 2010/2009 team. So we can look into improving that project. What we will be further researching: PUF project – Angela, Divya, Asha Plastic degradation – Adi, Uros, Fatty acid producing e. coli – Anthony, Isiah E. coli waste management – Cara, Bob The next time these ideas are presented – genes and a schematic should be presented. Next weeks: - April 10 –meeting with Dr. Robinson - April 15 – general meeting to prepare for the second round of advisor’s presentations - April 17 – advisor’s meeting for second round of presentations - April 22 – general meeting for the third round of presentations - April 24 – advisor’s meeting for 3rd round, entrepreneurship and human practices proposals - April 29 – quick update to everyone and a quick summer plan - May 1 – last meeting! The advisor’s meeting to give updates and feedback on our main and side project. Entrepreneurship and human practices will also be discussed. This is when we will plan our summer schedule and agenda.
iGEM Saturday meeting 4/14/12 Project Palooza! Microbial Expression and Characterization of a Sequence-specific RNA-binding Protein for Translational Regulation - Express PUF in prokaryotic cells and turn it into a biobrick - Can we use PUF as a translational repressor? This would allow for a more rapid change in protein levels. - Using PUF is a relatively new idea. We would need to study how good the affinity of PUF proteins are. (Binding works so that 2 amino acids interact with 1 nucleotide. A 16 amino acid PUF protein recognizes 8 nucleotide sequence.) - Characterization: Artificial 8 base pair design for inhibition of gene expression. - Optomization: Make a control, and then one nucleotide sequence with 1 one bair pair off. Study the affinity of PUF to both sequences. - Biobricks: 1 biobrick for each of the PUF repeats (8 in total), multiple recognition sites, modular devise for translational regulation. - This is a significant project, but need to look into applications. That just makes it that much cooler. Compile ideas and send to Angela by Monday night. Resveratrol and Piceatannol Project (Cara’s new project) - Resveratrol is turned into piceatannol in the body, which then goes and alters genes expressions when fat cells mature. It interacts with the insulin receptors on immature fat cells, stopping them from maturing. - 1) Can build piceatannol from the ground up (that’s hard.) 2) Use E. Coli to metabolized piceatannol from wine/grapes/etc (but how much is actually there? Is it actually efficient to do this?) 3) Put resveratrol in a medium, then use E. coli that would digest it into piceatannol and secrete it. o #1 and #3 have both been done. - Going forward: Look at stability/solubility/degradation. Apparently piceatannol also inhibits something in humans? What part of piceatannol is actually interacting with the insulin receptor? (To check on this last one, contact the Theorectal Biophysics group on campus to see if we can get time on their computers to do some protein docking modeling.) Edit: An email update on this has been sent out. - Application idea: Synthesize piceatannol (somehow) so that it will work in the body and be able to help maintain a healthy level of body fat. Create an E. Coli system that will allow piceatannol to work in the human body.
iGEM weekly meeting 4/15/12 Announcements Bob – Making progress to learn wiki coding and editing. Will also try to edit the wiki with current ideas and team info. Adi – meeting with Joe Bradley, Angela’s contact, as a potential Entrepreneurship advisor. Meeting is at Friday at 1:30. Angela will also be attending. Dr. Amos has also given card access to DCL to Adi, Uros, and Angela. They also received lab tours and saw the technology we now have access to. Adi is working with Alex to see how quickly the website can be up and running (ideal – end of the month). Also has some contacts if we decide that the PUF application should be HIV. UNC Prof. Tracie Hall will be contacted to see if PUF has uses with HIV. (Contact by Angela) Uros - Prof. Ha was contacted over PUF research. Emails have been gathered and professors have been contacted over potential collaborations. All of the labs have been quite friendly and receptive. The more opinions the better! Anthony – Checking with Courtney and Melissa that the books are balanced. The traveling funds we applied for are the 3 days the Friday weekend of July 13-15. (Leave Friday, get back on Sunday). Busing and driving are being considered as options. Asha – will be visiting the Engineering Council office to check out the availability of funds for the July traveling. Also, the U Mich pres will be emailed about human practices collaborations. Angela – Next Sunday the President of the University of Michigan iGEM will be coming to our meeting! Contacted Mat Song, last year’s human practices director, and that correspondence has been sent to Asha. Divya – Undergraduate Research Symposium went well! The pictures from that will be posted to facebook, and a new twitter and flicker will be created for iGEM to broadcast our work. During the summer, we will also have a blog on the iGEM website, spearheaded by Divya and Cara. Divya will also be creating a biography of our work, focusing on our opinions and perceptions of synbio before, during, and after the summer. Everyone is invited to the IGB Fellows, but previous members will be presenting the old E. Chiver poster. Everyone is registered though, so go grab a free lunch and listen to the talk! Cara – Let’s have a get together before finals! The tentative date is reading day. This will probably be our last meeting/get together before dispersing for finals and until May 15th. Agenda What’s our timeline? Proposed timeline: Next Tuesday – advisor’s meeting to present our more specific project proposals. Human practices and entrepreneurship should also prepare presentations. April 22 – presentations to update and further refine proposals. April 24- Advisor’s meeting: Human practices and entrepreneurship give formal presentations to advisors. May 1 – last advisor’s meeting to discuss final plans for the summer and for projects. This is the last meeting before dispersing! (In general, weekly meetings are updating and planning the projects. Advisor’s meetings are for presenting and getting critiques on these project ideas and plans). More specific plans: - Phat project: A solid plan will be developed by the last advisor’s meeting. So this project will lag behind the PUF project slightly. Phat group will physically meet Monday night at the UGL. - Human Practices: We will go ahead with the idea of standardizing the search for biobricks and past iGEM projects. - Entrepreneurship: Progress does depend on what the final project will be. At that point the entrepreneurship advisors will helps us make a decision about what plan of action is best for the entrepreneurship competition. Once the project is known and the option is chosen, the application will be a relatively simple execution. Major project should be made during the summer. - Wiki: Does a separate wiki need to be done for the entrepreneurship competition? The wiki template will be set up, so that during the summer our data will just be slotted in. This minimizes the work done during summer. - PUF: further applications will be looked into. We will google + hangout on Monday night to discuss the proposal presentation. Take the initiative to contact UNC researchers if necessary. Next advisor’s meeting! Dress up to take a formal picture (that means suits and jackets!) Tentative agenda: Brief discussion of ideas for entrepreneurship and human practices. Then PUF and Phat present, followed by a great deal of discussion. We will also ask if it is feasible to do a main project, a side project, and the entrepreneurship competition.
iGEM Advisor’s Meeting 4/17/12 Project Proposals Round 2 Resveratrol Metabolite Blocks Apidogenesis - Why aren’t we just producing lots of resveratrol and putting it in the body? The body will naturally change it into piceatannol for us. Response: Does it happen in the necessary quantity and concentration? We want to make these molecules more stable and more soluble in the body. - Resveratrol is widely regarded as a wonderful compound to extend life and help health. It’s widely researched and popular, but there is much controversy about it still. - How are we going to make this more soluble? Add groups to the non-active sites of the molecule. Groups increase solubility, like halogenation. - Do these modifications change the molecule itself? Well we can run computational tests on that. We can also biologically make resveratrol derivatives and test them like a pharmaceutical company. o Beyond looking a solubility, computational testing might not be of much help. You really need to synthesize libraries of the molecule and test them. - Need to look into P450 enzymes. But we want to stay away from protein engineering. We can buy and test a range of enzymes though. - Computational side? The molecule docking technology to do simple hydrogen bond analysis is free, but for the more complex and detailed information we need an expert. Courtney will consult with one person in her lab who has experience in this area. o The computation sounds like it is beyond the scope of the summer. o Our best bet in terms of time is to just make a few analogs and test them. Microbial Expression and - PUF works with only 8 base pairs of recognition. - Where should the PUF binding site go? Let’s test a bunch of models with it in various places to see which is the most effective (via visibility of YFP). - Need to clearly understand the PUF mechanism. This ensures that it is binding to a specific RNA sequences and not just the PolyA tail. - Applications: the PUF library is obvious and useful, but could also use PUF as a scaffolding tool in the style of zinc fingers. - How about engineering different kinds of PUF? First need to make sure it works in prokaryotes, then test the first type of PUF and optimize where the binding site should be. Then and only then we should look at making different versions of PUF and starting the library. - For this to work at all, it is necessary to make really sure that PUF works in E. coli. o It is possible to make this work with cDNA. Are we sure that we can get the cDNA from UNC? - It is a small protein. It might be easier to just synthesize it rather than clone it. We should definitely start with cDNA.
iGEM Weekly Meeting Minutes 4/22/12 Announcements Angela – New advisors! Kori Dunn, Angela’s graduate advisor in Prof. Rao’s lab. Prof. Joe Bradley of Business and Administration is our new Entrepreneurship advisor. Everyone needs to sign up for the iGEM dropbox, there are important papers and documents there that are useful for everyone. We will video chat with Dr. Wong will happen this Tuesday from 3-4 at the 2nd floor of the gatehouse. If the advisor’s are available, we will have a normal advisor’s meeting from 4-5 (this is tentative, watch for a confirmation email). Angela has received the PUF plasmid from UNC and is looking to see if the transformation is successful. If the transformation is successful we will move onto designing the construct. University of Michigan iGEM president is coming down this week. When will we meet with him? Isiah – not present, but everyone needs to send him the categorized inventory information! Adi – Met with Joe Bradley. PUF and Phat projects were discussed, but mainly PUF. If the PUF project is successful, we will have made steps towards making a toolkit. We have the option of marketing our toolkit or targeting a specific disease. Our first step is intellectual property. The patent database, USPTO, and Espacenet will be searched to see if any patents exist already. If we choose the business plan model, we will market our team as though we are a company. We will discuss how we will start and grown our company. Divya – An email has been sent out about the IGB symposium. Last year’s project will be presented at the poster part of the day by 2 former iGEM members. If you want free breakfast and lunch, register! Our theme is “other IGB.” The brochure will be further discussed with Melissa. Asha – The final Engineering Council meeting of the year is next Wednesday, and the retroactive summer travel funding will be discussed. Human practices has contacted Northwestsern, Brown-Stanford, and University of Michigan to collaborate. Instead of building a whole new website, Angela will contact Alyssa (Cornell) who is an administrator of the Alumni iGEM site to see if we can create a new page on their website. The point is to collaborate, not compete. In the fall we will also look into potentially volunteering to teach middle schoolers/ high schoolers about synthetic biology. Bob – Will be working to put all of the meeting minutes up on the wiki. The website looks basically the same as last time, but David has been contacted about how to make the wiki look more like a website through templates. Alex and Bob will share contact information to make everything look awesome. We also want to put our ideas on the wiki so that we can have them available for everyone to see the time-stamp of when we came up with it. Anthony – Estimated budget to travel to North Carolina for next year’s IBE conference. Cara – There is concern that the proposed phat project is too roundabout. A better idea might be to simply mutate the cytochrome enzyme that converts resveratrol to picetannol by sending it out to a company and then just testing all of the different versions. Tentatively, it sounds like this is a long term project. It would be better to make 1 of 2 simple constructs to test. Social stuff: We will have dinner on Reading Day together. Go to one place for actual food, then after to a dessert place! Will’s comments – As an RSO we can set up our own poster session about our iGEM projects and synthetic biology. We could do it at the end of the fall semester and invite other research groups who would like to present at the end of fall semester (there aren’t many symposiums at that time.) Agenda Bootcamp: - Are we going to be ready to go into lab? Who is good at what? We need to start thinking about how we are going to divvy up lab work. - We need to write out specific protocols. - We will partner up. - We will set aside time at the beginning of the summer to simply write out protocols and seriously and thoroughly learn all of the procedures. - Overall, we need a good wetlab plan as soon as possible. PUF stuff : - The PUF sequences have been put in the iGEM dropbox. - Why do we need the PUF sequences? Because we need to design primers. o Yellow = 8 PUF repeats o We got this from UniProt, searching for PUF1. The information from this site has been put on the Word File in the dropbox. o An alternative search site is the NCBI, using search “Protein”. - Biobrick foundation website has lots of assembly methods. Using RFC, we get the biobrick prefix and suffix for the PUF coding sequence. These prefix/suffixes contains restriction sites. *****put website from the green handout here ******** - Plasmid backbone: pSB1c3 (this is what we submit our biobrick in). pSB=plasmid synthetic biology. C= colminithol resistance. A map of this backbone can be found on the parts registry to physically see the restriction sites. - Annotation is getting sent to everyone.
iGEM Advisor’s Meeting 4/24/12 Conference Call with Dr. Zefeng Wang_of UNC___ - Slide #2 – 2 constructs (one with PUF binding, one without). The one without the PUF binding site gives YFP because it can be translated. Dr. Wang wonders if this will this work? - Currently there are no papers about the prokaryotic recognition sequences. Dr. Wang thinks it will not work. He recommends changing to a reporter that will work. - Papers suggest that PUF binds to the 3’ UTR in eukaryotic cells. We are unsure of if this will work in prokaryotic cells. No one knows exactly how PUF works in eukaryotes – they recruit other factors that stop translation. These other factors might not exist in prokaryotes. - There is a paper that claims PUF has worked in prokaryotic cells, but no plasmid was included in that paper. - Yeast 3 hybrid system works well in Dr. Wang’s lab. He can provide this as a modular system to start with. This saves us the trouble of what if the recruiter doesn’t work. - Many PUF mutants can be nicely expressed in E. Coli. Some cannot be expressed. - We can also try ASRE – artificial site specific RNA endonuclease. But we wanted to do it in E. Coli. - Dr. Wang ran an experiment where PUF was used to repress Bgalactosidase. The PUF binding occurred in the gene. The lower expression is from RNA degradation. PUF doesn’t cut RNA, just bind it. Dr. Wang put the PUF together with an endonuclease that cut the RNA when PUF bound to it. But that is not PUF anymore, it’s ASRE (the name of the fusion protein). - The whole ASRE complex looks like: Flag-PUF-linker-endo - Dr. Wang’s lab has a library of these ASRE’s that they are willing to share with us. But the paper is unpublished, so the data (if it works) needs to be treated confidentially. We would not release the information to the iGEM website until everything is collected and we have conveyed the results to Dr. Wang. - Complications: We don’t know what the rate-limiting step yet? The binding or the cut? Change design so that PUF binding site is between the RBS and the gene. We put the PUF binding site multiple times to try to increase the effectiveness. Could rearrange so that the reporter is under the repressor. - Distance between binding and digestions sites is very close. - To add multiple binding sites, where would they go? Can we add one to the 3’ UTR end so that it’s after the stop codon? Does it work? - Possible death mechanism: Engineer a PUF between the start codon and the promoter. It’s a highly conserved region so it will stop the manufacture of many proteins. - We can try multiple scenarios. We want to use the existing protein and change the binding site, as opposed to using mutated PUFs. - Dr. Wang’s overriding concern is if/how the reporter will work. Is there any natural repressor in the protein? Could use Lac I and B-galactosidase. Want to arrange it so that we control gene A, gene A and YFP have inverse expression. Once this works, changing modules will be easy. We will investigate more cases like Lac I. - Dr. Wang already sent the construct. But Dr. Wang will look into sending and MTA over to Dr. Jin. - Dr. Wang’s lab has every possible ASRE in a library. This is a good thing to look into further down the road. Then all we would do is test the readout from the different ASRE. - Question about plasmid Erin sent: It is for eukaryotic expression but it does replicate in E. Coli. It is PUF with another fusion that changes splicing in eukaryotic cells. But now we need the new plasmid with the fusion protein ASRE. - Erin used a step-wise amplification system to generate the library of ASRE. You can make it one-by-one, but the step-wise PCR created (in theory) all possible PUF. - Dr. Wang is open to collaboration and will share needed plasmids with us. - Antibiotic resistance will be Amp (maybe K? depends on which backbone). - The current backbone has K resistance. 3 plasmids: reporter gene with YFP that is controlled by inhibitor protein. Gene 2=codes for inhibitor protein and has PUF binding site. - 3 markers should be enough. Although we can combine to get just 2 plasmids. Discussion - It is clear that binding is not enough. We need the PUF binding and endonuclease activity. Dr. Wang’s lab will send us their contruct. - The concern right now is the location of the binding site. Can we choose one or can we engineer one? - There are many types of repressors to choose. We need to research which would be the best choice. For next week we will remodel the construct and find different repressors. Also look for a biobrick part. - It’s a big help that they already have the whole PUF library. - For the RBS we will use the Biobrick. This is because we want to submit a standard part, so we should standardize everything. Because we have to ligate everything, it’s a problematic idea to put the binding site between the RBS and the gene. - Other idea: Remove the loop area from the end of the mRNA. The 3’ UTR is always involved in stability, so if our PUF binds on the loop it would be more susceptible to the nuclease. Cara’s phat project - Buy resveratrol and a cytochrome P450 (several have been found, a human one and a mutant Bacillus one. However the Bacillus one may not be available for purchase online.) Sigma Aldrich is our potential vendor. - Resveratrol ranges in price and available styles (approx $100 for 100 mg, approx $300 for 500 mg) - The goal is to create mutant piceatannols. So we mutate the cytochrome P450 to see if we can get mutant piceatannols. - It’s hard to work with cytochrome P450 in the prokaryotic system because it’s made to work in eukaryotes. That’s why we can the bacillus cytochrome. - Three available ways to make mutant enzymes: site specific mutagenesis by a company, arichrome PCR, chemical mutagenesis - Overall idea for a lab plan: We put the mutated cytochrome in the e. coli and feed it resveratrol. We get a chip that binds to the insulin receptor and see what has binded to our chip to see what is happening. - Is this too ambitious? Or is it totally possible? There are conflicting opinions. - Biggest problem is the low solubility. So all of the assays and screenings are not easy. - Our ideal mutant would be more soluble, because then it would avoid degradation is the bloodstream. This random mutagenesis is a bit of a game of luck. How many mutations do we need to go through before we find something good? How many residues are there? - If we have directed group addition or the making of derivatives, it might be better and faster and potentially yield better results for our mutated piceatannols. - Feedback inhibition occurs when resveratrol and piceatannol inhibit the cytochrome that creates the mutants. - The painful part is where we need to evaluate each clone by an individual assay. Is there a way to create a florescence detection method? This is much easier. That way if the chemical is modified we see a color. Then we can do a ton of wells and only examine the wells that have changed color. - What is our desired endpoint? We don’t know what our final product A is. We’re just looking for the best product, so how can we know for sure? Extensive testing is needed. There are 2 unknowns (the enzyme and the compound.) - This had morphed into a pharmaceutical project, which is time consuming. - Still a good side project if we can obtain and test various derivatives. - Can we get constructs from people who have published? Then we can make sure that it will actually work as expected in E. coli. - 2 important things: We made a novel chemical and we made it in a commercially viable amount (g/L). We need to engineer the cell to produce it in proper amounts. So look at making P450 more stable so it can produce more. - Purdue paper: They did their research in cell cultures, but mentioned degradation in the body. So does it degrade in the cell culture? Announcements -New advisors: Kori Dunn (3rd year graduate student from Prof. Rao’s lab), Amet (also a 3rd year grad student from Prof. Rao’s lab), Prof. Joe Bradley for Entrepreneurship. Agenda for next weekly meeting - We are wrapping up for summer next week! It is important that we make the most of our meeting time, so come on time and come prepared! - Sunday Meeting agenda: 1) Angela finishes up the biobrick talk 2) Vote on projects. Do we want 2 equal projects? A main project and a side project? Which one is which? 3) Vote on bootcamp. Do we want a bootcamp? Will we split into pairs? How long will it last and what lab procedures will we cover? 4) Create a solid wetlab plan. Make one for any and all projects that we have. Start simply yet thoroughly and let the gold medal guildelines determine our progress. Make sure everyone is clear on this plan and understands all of the constitutive components. 5) Vote on vacation time. When will we reconvene for summer? Before coming back to work, what should everyone have done? (Reading papers, awareness of lab procedures, just take a brain break). 6) If time permits, general announcements, including an update on reading day’s social outing.
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! =)
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.
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.
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.
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.
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. -
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.
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.
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.
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.
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.
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.
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.
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 =(
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!!
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.
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!
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.
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.
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.
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.
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.
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.
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
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.
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
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
"
