Part:BBa_K2933240

RBS a+Linker g+GST+Linker e+BlaB-14

This part consists of RBS a, protein coding sequence(GST+Linker e+BlaB-14), the RBS and the protein coding sequence can be connected by linker g. 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1287
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 113
  

Usage and Biology

This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein BlaB-14. It encodes a protein which is BlaB-14 fused with GST tag. The fusion protein is about 54.3 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of BlaB-14 and combine the two parts with the cutting site of Prescission Protease. 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

First, 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.We then extracted plasmids and performed double enzyme digestion verification.

Figure 1. (a) The PCR result of BlaB-14. (b) 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

[1]González LJ, Vila AJ. Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-β-lactamase BlaB. Antimicrob Agents Chemother. 2012;56(4):1686–1692. doi:10.1128/AAC.05835-11
[2]Yum, J.H., Lee, E.Y., Hur, SH. et al. J Microbiol. (2010) 48:358.https://doi.org/10.1007/s12275-010-9308-5