Part:BBa_K2933258

RBS b+Linker h+His+Linker a+Sumo+Linker b+VIM-66+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+VIM-66) 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
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 298
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 298
  Illegal NheI site found at 75
  Illegal NheI site found at 1206
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 298
  Illegal BglII site found at 187
  Illegal BglII site found at 1135
  Illegal BamHI site found at 386
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 298
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 298
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein VIM-66 and T7 terminator. It encodes a protein which is VIM-66 fused with His-Sumo tag. The fusion protein is about 40.3 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

we used the vector pET-28bs 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 result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification

References

[1]Yoshihiro Yamaguchi. Wanchun Jin. Kazuyo Matsunaga. Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor. J. Med. Chem.200750266647-6653

[2]Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.

[3]Christopeit T , Carlsen T J , Helland R , et al. Discovery of novel inhibitor scaffolds against the metallo-β-lactamase VIM-2 by SPR based fragment screening[J]. Journal of Medicinal Chemistry, 2015:151017114758002.

[4]Christopeit T , Yang K W , Yang S K , et al. The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor[J]. 2016.