Difference between revisions of "Part:BBa K2933182"

Latest revision as of 11:43, 24 September 2019

T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+NDM-23+T7 terminator

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+NDM-23)and the biological module can be built into E.coil for protein expression.

Sequence and Features

Usage and Biology

This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of NDM23 and T7 terminator.It encodes a protein which is NDM-23 fused with His and Sumo tag. The fusion protein is about 54.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of NDM-23 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

We used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

References

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