Part:BBa_K2933282

RBS+Linker h+His+Linker f+BlaB-14+T7 terminator

This part consists of RBS, Linker h, protein coding sequence(His+Linker f+BlaB-14) 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

Assembly Compatibility:

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

  Illegal NheI site found at 93
  Illegal NheI site found at 883
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 689
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part is made up with six basic parts(RBS , Linker h, His, Linker f , BlaB-14 and T7 terminator ). It encodes a protein which is BlaB-14 fused with His tag. The fusion protein is about 28.3 kD. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28a 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