Week 8

8/12/13
The Tecan run stopped after cycle 2 due to an error, so we weren’t able to further characterize K598010 and K750000 in the DH10B cells. We decided to redo the Tecan run by growing up some leftover cultures, but they ended up growing very slowly. We decided to instead just start new cultures from the glycerol stocks and restart the Tecan run tomorrow. In addition, we ran several different PCR reactions for our TAL repressors with different polymerases to see if it would result in our amplified product of interest. According to our sequence results of pSB3K3, the site-directed mutagenesis was successful and now the plasmid no longer has a BsaI site. We began amplifying pSB3K3 for Golden Gate Assembly as well. After running our PCR products on a gel, we are finally getting the correct bands for the TAL repressors; however, there is a certain level of streakiness in each lane. The amplified product for pSB3K3 did not show any bands.

8/13/13
We ran another Tecan trial involving K598010 and K750000 with our new strain and will analyze the data tomorrow. In addition, we ran several different PCR reactions involving our TAL repressors and pSB3K3. We decided to lower the template concentration for the TAL repressors in order to address the issue of streakiness. In addition, we ran a single reaction with no polymerase to see if there would still be streakiness without the polymerases. The streakiness was still present in all of the lanes, except the lane with no polymerase. This leads us to believe that there might be nonspecific annealing during PCR. We also chose to amplify pSB3K3 with Taq and Pfu instead of Phusion polymerase. Each of these tubes had an annealing temperature at 64, 60, or 55 degrees C. We also began the initial modeling of our construct with MATLAB and continued updating our wiki.

8/14/13
Today we had a NorCal iGEM team meetup here at Davis, where we met up with UC Berkeley, Stanford-Brown, and UCSF to discuss our projects in further detail. We each received valuable feedback on our projects and ended the day with a round of bowling. After everyone had left, we headed back to the lab. We decided to combine all of the PCR reactions that had worked for the two TAL repressors, run them in a gel, and then purify the final product. We also did a PCR clean up on the pSB3K3 PCR product and checked on the Tecan and realized additional time was needed to finish the run.

8/15/13
We are still waiting for our oligos to come in for further Golden Gate Assembly. We measured the DNA concentrations for our two Golden Gate-ready TAL repressors and analyzed some of the data involving K750000 and K598010 from the Tecan. The data seemed to show that the presence of arabinose or theophylline did not have a significant effect on overall fluorescence. We believe that DH10B may have heightened expression of AraC that results in greater repression of GFP. For most of the day, we either worked on modeling or updating the Wiki. We hope to start doing Golden Gate Assembly tomorrow.

8/16/13
We received the oligos necessary for Golden Gate Assembly, but realized that the larger oligos were delivered in amounts of 200 nanograms. We decided that we should amplify the products, so we ordered more primers for further PCR of these oligos. We hope to get them by Wednesday at the latest. We continued updating the Wiki and designed primers for sequencing of our final construct.