Difference between revisions of "Part:BBa K2933256"

Latest revision as of 15:00, 25 September 2019

RBS b+Linker h+His+Linker a+Sumo+Linker b+BcII-194+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+BcII-194) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features

Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein BcII-194 and T7 terminator. It encodes a protein which is BcII-194 fused with His-Sumo tag. The fusion protein is about 40.1 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First,we obtained BcII by PCR.

Figure 1. The PCR result of BcII.
Then we used the vector PET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Figure 2. Left:The plasmid of BcII.Right:The verification results by enzyme digestion.

References

[1]Sung-Kun Kim, Mara Demuth, Sara R. Schlesinger, Sung Joon Kim, Jonathan Urbanczyk, Robert W. Shaw & Hyunshun Shin (2016) Inhibition of Bacillusanthracis metallo-β- lactamase by compounds with hydroxamic acid functionality, Journal of Enzyme Inhibition and Medicinal Chemistry, 31:sup4, 132-137, DOI: 10.1080/14756366.2016.1222580