Week 5

7/22/13
We ran two different restriction digests for BBa_K750000 and pSB3K3 as part of our protocol for site-directed mutagenesis by PCR. According to the restriction digest, there were no bands present in the lanes containing the pSB3K3 digest. There were bands of appropriate size for BBa_K750000. We decided to transform the DNA within the restriction digest anyways. We also measured the DNA concentrations for the two TAL repressors. The transformations of BBa_K598010 and pSB3K3 were successful and were inoculated in LB for further culturing. We ordered primers for GFP, TAL repressors, and our two different plasmid backbones, which will be used in Golden Gate Assembly. We also cultured a strain of E. coli, MG1665Z1 that endogenously expresses LacI, TetR, and AraC. We will use this for our construct.

7/23/13
Our overnight cultures for BBa_K598010, pSB3K3 and MG1665Z1 all looked good. A portion of the cultures were saved as glycerol stocks, and BBa_K598010 and pSB3K3 were miniprepped. The transformation of the mutant BBa_K750000 was successful and was inoculated in LB for further culturing. However, the transformation of the mutated pSB3K3 was not successful, which was reasonable considering the lack of any bands on the gel we ran yesterday. In light of this failure, we realized that pSB3K3 actually had an insert part that we neglected to take into account for in our site-directed mutagenesis reaction which we corrected and repeated today for both the previous miniprep and the one that we performed today. We also began making MG1665Z1 competent for future use in our constructs.

7/24/13
We sent in the mutant DNA miniprep of BBa_K750000 for sequencing to see if our site-directed mutagenesis actually worked. The transformations of the mutant pSB3K3 were successful, so we started one LB culture to grow overnight. We made new competent cells with MG1665Z1, so that we could use them for future constructs. We are testing our new competent cells by transforming them with mutant BBa_K750000 and BBa_K598010. In addition, we realized that we were running out of LB and decided to make an additional batch for the lab along with additional LB plates containing ampicillin.

7/25/13
The newly made competent cells were successful in taking up DNA for BBa_K750000 and BBa_K598010. Both were cultured in LB for us to use as a test for induction and GFP measurements tomorrow. The site-directed mutagenesis for BBa_K750000 was also successful, so there is no longer a BsaI site in the sequencing results. We did a miniprep of the mutant pSB3K3 and plan to send the plasmid out for sequencing when primers have been ordered. However, we may not use pSB3K3 until after we have created our entire construct on one plasmid and then we shall decide to vary copy number. After looking at the primers that were used for site-directed mutagenesis, we realized that they were targeting a false BsaI site. Therefore, any of the work done with pSB3K3 would have to be done with newly ordered primers that will hopefully be received by next week.

7/26/13
We made stock solutions of arabinose and theophylline for future testing. The cultures containing BBa_K750000 and BBa_K598010 were too turbid for Tecan testing, so we grew out the cultures to ideal optical density. After growing our cultures to an OD600 of 0.5, we tried out a TECAN test trial with BBa_K750000 and BBa_K598010. We aliquoted varying concentrations of theophylline and arabinose and decided to run a trial over night to test the behavior of these two constructs. In our spare time, we learned more about HTML5 for updating our Wiki.