Part:BBa_K2933196

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

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+VIM-66)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 1298
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 390
  Illegal BglII site found at 279
  Illegal BglII site found at 1227
  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 VIM-66 and T7 terminator.It encodes a protein which is VIM-66 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 VIM-66 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 insert VIM-66 gene into the standard vector then transfer it into E.coli.

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.