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Team:UC Davis/Notebook
From 2012.igem.org
Week 1
Re-hydrated Parts to get practice: J23101 E0240 E0040 R0010 pSB1C3 pSB1A3 pSB1AK3 pSB1K3 B0034 I13602
Transformed these parts into E. coli strain DH5α
We cultured 4 parts from glycerol stocks for more practice! J45120 for wintergreen smell! In 2mM salisylic acid. J45200 for banana smell! In 5mM isoamyl alcohol. R0010 for it's promoting abilities. E0240 for GFP! We also narrowed our project down to three ideas: Producing spider silk proteins in bacteria. Degrading plastics in bacteria. Creating an Archaea toolkit and using them for bioplastic, biofuel, and isoprenoid production.
Tuesday June 19th
Smelled banana scented E. coli. Miniprepped the R0010 and E0240 liquid cultures. Messed up the protocol twice due to inexperience, and got to practice DNA purifications! Nanodropped the miniprep samples to teach people how to use this instrument. Made liquid cultures from our colonies from yesterday's transformation. Set up a digestion of the R0010 and E0240 parts. (Cut R0010 at SpeI and PstI sites; cut E0240 at XbaI and PstI). Made a small 1% agarose gel. Made plates and LB media. Read papers on spider silk and plastic degradation
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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