Team:WashU StLouis/Parts

From 2014.igem.org

Biobrick Cloning
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 The original idea was to break the nif cluster from Cyanothece sp. 51142 into six different parts since the entire cluster is upwards of 35 kb. Primers were designed, and the desired fragments were produced via PCR. After running a gel to confirm the products, it was observed that some of the PCR reactions had not produced any of the desired fragments. Using a temperature gradient and DMSO, all of the fragments were eventually obtained. The BioBrick backbone pSC1B3 was isolated from an existing BioBrick part taken from the distribution plate using PCR and the codes for the BioBrick prefix and suffix as primers. This successfully produced the pSC1B3 plasmid, into which the six different fragments were inserted using Andrew Ng’s CPEC protocol. The now complete plasmids, backbone plus insert, were transformed into the E. coli strain XL1 Blue and plated on LB plates. The E. coli were cultured overnight, and four cultures from each plate were selected for colony PCR the next morning. The colony PCR indicated some positive results, and the plasmids were extracted from those strains, digested with the enzymes XbaI and SpeI, and confirmed with a gel. Only two of the BioBricks showed correct bands from the enzyme digest. After all of this was complete, it was discovered that all of our prospective BioBricks including the two successful ones, all contained one or more illegal restriction sites, thus invalidating their submission.
Submitted Biobricks: BBa_K1385000: CpcG2 promoter expressing TetR BBa_K1385001: Hybrid CpcG2 promoter expressing TetR
Parts Table
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<groupparts>iGEM014 WashU_StLouis</groupparts>

Team:WashU StLouis/Parts

From 2014.igem.org

Biobrick Cloning

Parts Table

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