Part:BBa_K2933253

RBS b+Linker h+His+Linker a+Sumo+Linker b+GIM-2+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+GIM-2) 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
  Illegal PstI site found at 1128
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 298
  Illegal NheI site found at 75
  Illegal NheI site found at 1158
  Illegal PstI site found at 1128
  
• 21
  INCOMPATIBLE WITH RFC[21]

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

  Illegal EcoRI site found at 298
  Illegal PstI site found at 1128
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 298
  Illegal PstI site found at 1128
  
• 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 GIM-2 and T7 terminator. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4 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.

Experimental results

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.

Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification

References

[1]Skagseth S , Akhter S , Paulsen M H , et al. Metallo-β-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding[J]. European Journal of Medicinal Chemistry, 2017, 135:159-173.

[2]Wendel AF, MacKenzie CR. 2015. Characterization of a novel metallo-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany. Antimicrob Agents Chemother 59:1824 –1825.

[3]Borra P S , Samuelsen O , Spencer J , et al. Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-beta-Lactamases[J]. Antimicrobial Agents and Chemotherapy, 2013, 57(2):848-854.

[4]Susann S, Trine J C, Gro Elin K B, James S, Ørjan S, Hanna-Kirsti S. L. Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1. Antimicrobial Agents and Chemotherapy Jan 2016, 60 (2) 990-1002