Team:UC Davis/Notebook/Week 6


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Week 6

BBa_K598010 performed in the Tecan as expected. With increasing concentrations of theophylline and arabinose, the fluorescence of the cells increased. Also, the addition of arabinose to wells containing liquid cultures of BBa_K750000 only showed a short spike in fluorescence that died out after about 2 hours due to the LVA degradation tag, and the addition of theophylline had no significant effect. Some of our primers have finally arrived, so we were able to start amplifying some of the parts we need for our construct that will be assembled through Golden Gate Assembly. So far we have started working on amplifying and adding BsaI restriction sites to the two TAL repressors, the GFP with an LVA tag and terminator from BBa_K750000 and the pSB1C3 backbone. We also took a soil sample to send to the University of Edinburgh for their project.

We ran a gel in the morning to test our Golden Gate PCR reactions. Two of the five reactions seemed to show the correct band sizes. We believe the failure of the three other reactions may be due to the low melting temperature (55º C). We decided to run the PCR reaction again with the melting temperature at 70º and 80º for the three unsuccessful runs. We also helped the Imperial College iGEM team by sequencing a construct related to our university’s project from last year. At the end of the day, we ran another gel to see if we could get the correct amplified product. Both of the PCR reactions with new melting temperatures at 70º C and 80º C seemed to not work and had irregular bands around 400 bp long. We saw these same bands in the our first Golden Gate PCR reaction.

In lieu of what has happened in the past two days, we decided to try seven different melting temperatures in an interval between 55º C and 75º C. We hope this will help us find the ideal melting temperature at which to amplify these Golden Gate products. We ran out of time towards the end of the day to run a gel and check the different PCR reactions. We plan on doing this tomorrow. We also spoke to a fellow UC Davis student about starting a synthetic biology club on campus. We brainstormed different ideas about how we would gain interest from the student body about such a club and believed that this could be great for human outreach.

Today we started off by running a large gel to check all 21 of our PCR products from yesterday. The two TAL repressors were not correctly amplified, which makes us question the design of our primers. In the majority of the lanes, the presence of aberrant bands at the 400 bp length may be evidence of mispriming. To mitigate this, we decided to redesign new primers. In the case regarding pSB1C3, the PCR amplification step was successful. We performed gel extraction and purification on the two GFP and the pSB1C3 PCR products in order to isolate our desired products from any side products or remaining template.

After obtaining very low DNA concentrations from the gel extraction of the pSB1C3 PCR product from yesterday, we decided to just simply do a PCR clean up on the remaining PCR products to get a better yield. So far, we have amplified product of pSB1C3 and two different GFP products with different flanking ends. We ordered new primers for the two TAL repressors and made sure there was no overlapping homology in the primers with the repeat domains of the TAL repressors. We received primers for our site-directed mutagenesis involving pSB3K3. We began the PCR reaction and hope to do a restriction digest next week. We also helped out a teacher at Davis High School by inoculating several LB cultures with cells expressing mRFP. The cultures will be miniprepped tomorrow for a transformation experiment. When we weren’t doing lab work, we were learning HTML for our wiki, which now has a Twitter feed.