Light Regulation Group - Benjamin Huang

Cloning the biobricks was more difficult than expected. Ben tried to do digestion/ligation using the restriction enzymes using the PSB1C3 backbone from BBa_K1017726, but transformation into E. coli strain DH10B yielded low efficiency with minimal number of colonies. He ran sequencing PCR with primers that bound to both the backbone and the target part. He found the prefix but not the suffix.

Ben and Cheryl tried different sequencing primers that bound to sites on the genes instead, and re-ran sequencing PCR to no avail. Next, they tried religating using higher molar ratios 3:1 vector to insert, and the sequencing PCR ran into the same issue.

We then got a new purified backbone from the distribution well from caroline and used that to clone. We also used Andrew's CPEC protocol to clone both the hybrid and normal part. We weren't able to clone the positive control because for the protocol you need 15-20 bp overlaps and the primers we had on hand did not allow for that. Also there was a very similar part on the parts registry so there was no use sending a part that essentially had the same Ptet and EYFP.

Using PCR purified products, the CPEC cloning had much higher efficiency than digestion/ligation. We ran colony PCR on the colonies, checking for both the prefix and suffix, and the colonies that came from the CPEC had the right bands. We sent in the sequences for sequence verification and successfully sent in 2 biobrick parts.

Biobrick Submission: 10/8/14

Nitrogenase Group - Caroline Focht