Part:BBa_K2933199

T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+BlaB-14+T7 terminator

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+BlaB-14)and the biological module can be built into E.coil for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 390
  Illegal XbaI site found at 47
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 390
  Illegal NheI site found at 167
  Illegal NheI site found at 1244
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 390
  Illegal BglII site found at 279
  Illegal BglII site found at 1050
  Illegal BamHI site found at 478
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 390
  Illegal XbaI site found at 47
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 390
  Illegal XbaI site found at 47
  
• 1000
  COMPATIBLE WITH RFC[1000]

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 BlaB-14 and T7 terminator.It encodes a protein which is BlaB-14 fused with His and Sumo tag. The fusion protein is about 40.3 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of BlaB-14 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

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

Figure 1. The PCR result of BlaB-14.

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