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From 2012.igem.org

Monday

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