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Team:UC Davis/Notebook
From 2012.igem.org
Week 1
Today was the first day of out iGEM team officially meeting in the summer. We got a look around the laboratory space we would be using, and since almost everyone on the team is new to iGEM, we went over the things we had to accomplish over summer. We re-hydrated some parts from the distribution kits to get practice, and those parts were: J23101 E0240 E0040 R0010 pSB1C3 pSB1A3 pSB1AK3 pSB1K3 B0034 I13602
These parts were later transformed into the E. Coli strain DH5α.
We cultured 4 parts from the glycerol stocks for more practice. The parts were: J45120, which emits a wintergreen scent. This was done in 2mM salisylic acid. J45200, which emits a banana scent, respectively done in 5mM isoamyl alcohol. R0010, as a promoter E0240, or more commonly known as GFP! Our team took time to discuss our project, and comparing our interest with the research that has been conducted thus far, we narrowed our project down to three ideas: 1. Producing spider silk proteins in bacteria. 2. Degrading plastics in bacteria. 3. Creating an Archaea toolkit and using them for bioplastic, biofuel, and isoprenoid production. We realized that more research had to be done to understand what each project specifically requires to judge how feasible and successful it could be.
Tuesday June 19th
We began the day with some wetlab work to keep on practicing techniques. The E. coli cultured with J45200 smelled like bananas, which was exciting! The R0010 and E0240 liquid cultures were miniprepped, but we messed up on them due to inexperience. More practice was done to learn the procedures of DNA purifications, while other members of the team nanodropped the miniprepped R0010 and E0240 liquid cultures for practice using the machine. Following the nanodrop, a digestion was set up of the R0010 and E0240 parts. R0010 was cut at SpeI and PstI sites; E0240 was cut at XbaI and PstI. We liquid cultured the transformations from yesterday, and made plates and LB media. In terms of our project, more research was done on spider silk and plastic degradation, as we read papers on studies that dealt with both themes and we looked at the methodology used to approach the problems, searching for similar assays and pathways that we could use.
Wednesday June 20th
As soon as we came into lab today, some of our team members began miniprepping the liquid cultures from yesterday. These were nanodropped, and the concentrations looked good! Some of our team members were also taught how to make and use gels, as we ran the digested parts from yesterday on a one percent agarose gel. We also made another stock of 1x TAE buffer from the 50x TAE buffer by dilution. Once extracted, the parts were ligated and transformed, which took some time. Once finished, we stored them for further examination. In terms of research, we spoke with one of our mentors, and narrowed down the possible project ideas to either plastic degradation or a toolkit for archaea. The spider silk seemed to be more and more impractical as we read more papers concerning the spinning and formation of the fibrous materials that make up spider silk.
Thursday June 21st
Today consisted of mainly reading and researching more into pathways for plastic degradation and meeting the gold medal requirements. The Ligation from yesterday turned out to be unsuccessful, but the parts we were trying to ligate were not immediately necessary, but it was rather done more for practice. We also went out as a team to a sushi buffet, continuing a tradition of team bonding on Thursday for lunch.
Friday June 22nd
Friday was assigned as a weekly meetup day with our entire team and our advisors. We explained where we are at in terms of researching for our project, and they gave us some interesting aspects we can look at. We narrowed our project down to just plastic degradation, as the archaea engineering would be a clear cut contribution to the parts registry, but would not be as interesting to the team as a whole and not a sufficient project to pursue for the entire summer. However, it was suggested that we make a toolkit for parts for archaea so future iGEM teams can pursue constructs in archaea.
Week 2
Monday signified the beginning of week two for iGEM! The day was mainly composed of reading papers and looking into ways to which we could degrade plastic. We also scheduled a visit to a local landfill, with an intent to see how the landfill deals with plastics and other synthetic polymers. The toolkit idea was also considered a secondary project idea.
Tuesday June 26th
We practiced lab protocols a bit more, and conducted more research after finding out the planned trip to the landfill. We also prepared questions and did research concerning what the landfill does and how we can benefit from it.
Wednesday June 27th
Today we went to the Yolo County Landfill to meet with Ramin Yazdani, the senior civil engineer at the landfill. We were given a tour of the landfill, and discussed procedures and systems Dr. Yazdani has in place regarding the decomposition of trash, specifically in terms of methanogens and methanotrophs. He similarly provided us with guidance concerning the direction of our project, and the possible application and impact our project could potentially have in terms of trash degradation.
Thursday June 28th
We spoke with our mentors regarding the feasibility of our project within the given time span. Ruled out polyurethane due to toxic byproducts.
Friday June 29th
Conducted more research in engineering methanogens and methanotrophs. We also looking into how our project could apply to the landfill, and discussed the feasibility of PET degradation versus methanotroph and methanogen consumption of methane.
Week 3
We further looked into the means by which our project can be outlined and planned. -Our team read a paper titled Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach to further understand the mechanism by which cutinase assists in plastic degradation. -We looked at the value of surface modification, specifically how it alters PET and whether or not it is valuable. -Research was conducted on PelB and the potential for a scar, and furthermore whether a scar will influence our results.
Tuesday July 3rd
Today we focused on wet-lab procedures -We rehydrated the PelB tag (J32015), T7 constitutive promoter (I719005), Stop (B0015), And LuxR inducible promoter (C0062). -We also transformed the parts mentioned above. TRANSFORMATION PROCEDURE HYPERLINK HERE. -We plated the parts. PROCUDURE HERE
Wednesday July 4th
We took the previous day's plates and ran liquid cultures on all of them. PROCUDURE -We took a half day for July 4th!
Thursday July 5th
We conducted a double digest of the T7 constitutive promoter (I719005) and Ribosome Binding Site (B0034). DOUBLE DIGEST PROCEDURE -We worked on the safety page of our wiki after concern arose regarding the safety of procedures in our planned project. We continued research regarding how we can plan and carry out our project.
Friday July 6th
We created an in-depth plan for how we were going to assess our project. We divided the project up into manageable sections and assigned particular areas to people on our team. -We took a look at the requirements for achieving the Gold medal, and wrote down and organized how we are going to achieve each criteria.
Week 4
We wanted to re-hydrate some parts, they are listed below. They were located in the distribution kits.
J23100 J23119 J23100 I13453 I13458 C0012 K206000
Along with these parts, we also looked at a new miniprep kit that was given to us for free, titled Biobasic. We wanted to test it in comparison to the Invitrogen kit we currently use. Following the hydration we transformed the parts and left them overnight in the 37 degree room.
Tuesday July 10th
Today we did a double digest of B0034 at the S and P sites and sequentially digested B0034 first at the E site using Buffer 4. We miniprepped samples using both the Invitrogen and Biobasic kits. Using the nanodrop spectroscopy, we determined that the Invitrogen kit was still better, so we continued to use that. We also began to look into Gibson assembly, and due to past iGEM inability to attain success, we requested the protocol from the iGEM Washington 2011 team.
Wednesday July 11th
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Thursday July 12th
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Friday July 13th
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Week 5
Our team was finally back together, as a majority of people were absent for the latter part of last week. A few of us went over what was done last week, and how there was an error in running the gel extractions, so we made a decision to re-attach the promoters to the B0034 RBS. Some of us began by running digestions of J23100, K206000, J23101, and R0010 at the E and S sites. B0034+C0012 was digested at X and P, J23101 was digested at S and P, and B0034 was digested at E and X. We also wanted to carry on some of the themes from last years project, and began trying to mutate K206000, or the pBAD promoter, to improve its function through random mutanogenesis. Some of our team members also began preparing the E. Coli strain, numbered MG1655, by streaking the organisms on plates from the glycerol stock. After, we rehydrated MG1655 strain. We also liquid cultured and plated DH5alpha cells and tried to make compotent cells for MG1655 strain, but put them in the 37 degree room. Some of our team members began to run error prone PCR (PROTOCOL) on the K206000 part as well, to make a part family as well. In our discussion of how we were going to assay our samples, we also ordered pNPB, another sample used to determine degredation in comparison to PET.
Tuesday
When the team came into lab today, we used the spectrophotometer and the OD600 readings were over 1, which signifies we overgrew them. The error came from placing the cells in the 37 degree room instead of at room tempurature. Oops. So we then remade the component cells for the MG1655 strain and extracted genomic DNA from the MG1655 and DH5alpha E. Coli strain. Luria Broth was made, and we incubated the starter culture and made inouetransformation buffer. We miniprepped the parts that were transformed yesterday, and sequentially digested B0034 and B0034+E0240 at the E site with plans for the X site to be digested on Wednesday. The PCR products from yesterday were ran on a gel to confirm the products, and then a purification was run, and the process was repeated twice.
Wednesday
Today genomic DNA from DH5alpha and MG1655 was purified and we restreaked DH5alpha cells from yesterdays sample. The purified PCR from yesterday was also put on a gel and extracted. Some of our team members also began working on the modeling of the cutinase enzyme using computer software to see how we could optimize degredation. More research was done in regards to making sure we had all the materials and that the assays to be conducted were also looked into and finalized. A gel was also ran of the purified MG1655 and DH5alpha genome.
Thursday
The day started off with us receiving the MG1655 strain we ordered from Yale University. Even though we already began to use samples from one of our mentors' labs, it was a good idea for us to make liquid cultures and plates of the cells in case something went wrong. The two enzymes required in the pathway for breaking down ethylene glycol also underwent PCR along with genomic data from the MG1655 and DH5alpha cells. Luria Broth (LB) was made for kanamycin plates as well. We preformed ligation and transformations of the genomic data into the DH5alpha cells, and successfully cultured competent cells. At the end of the day, we rehydrated primers for the mutated cutinase as well, with a hope that the mutations will improve the cutinase functioning. Friday
Week 6
Tuesday July 3rd
Wednesday July 4th
Thursday July 5th
Friday July 6th
Today was the day scheduled for the iGEM meetup! After doing some stuff in the lab (find out what everyone did), our team left around 1 P.M. for Genentech Hall at the UC San Francisco campus. There we interacted with teams from UC Berkeley, UCSF, and Stanford-Brown and presented our respective projects. The advice and critiques were helpful to us as it gave us a different viewpoint of how we can improve our project. We also socialized outside following the presentations, and it was an interesting experience for our team as a whole.
Week 7
More work was done in the soeing of DNA together while removing the restriction sites, however we ran into some errors while trying to correctly follow the procedure.
Tuesday July 3rd
The team split up again for more work and research. We finally got the strain we requested from Dr. Aguilar in Barcelona, and we also recieved the primers for the glycoaldehyde reductase compatability with an aerobic environment, so that was exciting. We similarly got a stock of ethylene glycol for which we can test the activity of the enzymes, and began culturing on it. We also continued our work on the SOEing procedure, trying to remove the Pstl restriction sites from the sequences after a brief mix up yesterday. We also got in contact with another professor here at UC Davis that does work in textiles and material sciences, and obtained some samples of PET that we can use in our assays of our enzyme.
Wednesday July 4th
Thursday July 5th
Friday July 6th
