Week 7

8/5/13
The minipreps containing BBa_J23101 were measured for DNA concentrations and aliquoted into the same test tube. These plasmids containing BBa_J23101 will be used by Davis High School for future transformation experiments. We spoke to our advisers in the morning and showed Dr. Siegel our preliminary data on our pBAD+Riboswitch+GFP(BBa_K598010) and pBAD+GFP(BBa_K750000) constructs that we had assayed a week earlier. He noticed that the output of the pBAD+GFP construct did not have a higher background level as time progressed. The lower background level suggests that after a certain amount of time the culture was degrading arabinose thus resulting in full repression of GFP expression. In order to mitigate this, Dr. Siegel advised us to use DH10B instead of MG1655Z1 for Tecan measurements. DH10B is known to not express the arabinose operon with the exception of araC. After finishing the restriction digest of pSB3K3, we ran the results through the gel and saw that there was no evidence of correctly sized bands. We decided to do a transformation anyways.

8/6/13
The transformation involving our potentially mutated pSB3K3 plasmid did not work, so we retried site-directed mutagenesis PCR, but increased the amount of template DNA in the reaction. After amplification and digestion, a gel was run to see if there was any evidence of an amplified product. It appeared that there was a band of the right size, which means the reaction was successful. At the end of the day, we did a transformation with the newly mutated pSB3K3 plasmid. We also tried ethanol precipitating some DNA in order to prepare it for electroporation.

8/7/13
The transformation involving the mutated pSB3K3 was unsuccessful. We redid the transformation with additional DNA. We also performed our first electroporation transformation with BBa_K750000 and BBa_K598010 into the DH10B strain. It took a little trial and error and the ethanol precipitations we did yesterday were not necessary after all, but hopefully it will be successful. We received new Golden Gate primers for the amplification of our TAL repressors with new BsaI sites. With the experience gained from difficulties with PCR in the past, we decided to try three different reverse primers on our two TAL repressors and then try different melting temperatures. The number of different combinations was a total of 30 different reactions. We hope that at least two reactions involving the two different TAL repressors will work. By working in parallel, we hope to reduce the amount of time that may be used in the future.

8/8/13
The transformations involving BBa_K750000, BBa_K598010, and the mutated version of pSB3K3 were all successful. We grew pSB3K3 in liquid LB. We plan on running a practice Tecan run involving BBa_K598010 and BBa_K750000 with our new strain DH10B. Tomorrow we will do a miniprep and sequence the plasmid for the miniprep. These will be cultured overnight. Unfortunately, all of the PCR amplifications of the TAL repressors were not successful. In light of these persistent issues with amplifying the TAL repressors, we are going to restriction digest them in order to confirm whether the DNA in our miniprep is does contain the TAL repressors that we want or just some contaminants. We continued updating our wiki with consideration of the project overview due date.

8/9/13
We ran a gel involving the restriction digest of the two TAL repressors. The bands appeared to be of the right size, which indicates that we do have the correct plasmid. We’ve decided to run another set of PCR reactions with additional template DNA and decided to extend the elongation time. We ran a gel of the PCR reactions to check to see if the correct product sizes were amplified. At the end of the day, we looked at our gel and noticed two different fragments in most lanes at the 600 and 800 base pair sizes. After talking to our adviser, we’ve decided to switch out our polymerase for an enzyme that can better handle a template with high G/C content. Both of our TAL repressors have approximately 67% G/C content, and we realized that this may be why we are only getting partially amplified product. We also realized that we were running low on DNA for additional PCR reactions, so we did another transformation with our two TAL repressors. We decided to grow the culture and do a miniprep over the weekend. The culture of the mutated pSB3K3 was miniprepped and sent out for sequencing. The cultures containing BBa_K750000 and BBa_K598010 were also grown up in LB until they reach an OD of 0.5. Then, the cultures were aliquoted into a 96-well plate for Tecan measurements. The part for Imperial College was also prepared to be sent overseas soon.