Difference between revisions of "Part:BBa K3182100"

Revision as of 16:21, 16 July 2019

pT7-CBDcipA-pCons-AsPink

AsPink dropout enabling colour-screening for positive colonies. Using BamHI and SpeI or PstI on both this part assembled in pSB1C3 and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein.

The part has a very strong expression with a T7 promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). This requires the T7-RNA-polymerase and bacteria with this should only be used after the C-terminal fusion protein has been assembled.

Figure 1. The first plasmid (left) contains pCons-AsPink which results in pink colonies. When BamHI is used together with either SpeI or PstI pCons-AsPink is cut out and replaced with a compatible biobrick such as an antimicrobial agent. When pCons-AsPink is successfully cleaved from the plasmid the colonies will be white. This results in a "pink-white screening".

Sequence and Features