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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
Miniprepped the liquid cultures from yesterday. Nanodropped the samples. Ran the digested parts from yesterday on the gel and extracted them. Ligated and transformed these parts. Made iGEM Contact List. Narrowed the project down to plastic degradation and Archaea.
Thursday June 21st
Ligation was unsuccessful Ate sushi buffet. Continued the tradition for team bonding.
Friday June 22nd
Met with the advisors. Narrowed down our project to one of plastic degradation. We also want to construct an Archaea tool kit. To be continued...
Week 2
Research. We ruled out the spider silk idea and decided to focus our attention on plastic degradation. 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 .
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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