Difference between revisions of "Part:BBa K2933133"

Revision as of 11:39, 25 September 2019

His+Linker a+Sumo+Linker b+BcII-194

This part encodes the fusion protein of His tag, sumo tag and BcII-194 to promote the expression and purification of target protein(BcII-194).

Sequence and Features

Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein BcII. It encodes a protein which is BcII fused with His-Sumo tag. The fusion protein is about 40.1kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of BcII and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

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

Molecular cloning

First,we obtained BcII by PCR.

Figure 1. The PCR result of BcII.
Then we used the vector pGEX-6p-1 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.

Expression and purification

Exploration of expression condition:
Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria. Test the OD600 number of bacteria, then pipe 5-10ul into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.Set the gradient of condition to explore how to express it best.We use 0.2mM IPTG, 16°C/0.2mM IPTG, 37°C/0.4mM IPTG, 16°C/0.4mM IPTG, 37°C/0.6mM IPTG, 16°C/0.6mM IPTG, 37°C/0.8mM IPTG, 16°C/0.8mM IPTG, 37°Cas different conditions.