Part:BBa_K2933262

RBS b+Linker h+His+Linker a+Sumo+Linker b+IMP-71+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+IMP-71) 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 1121
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 298
  Illegal NheI site found at 75
  Illegal NheI site found at 1146
  Illegal PstI site found at 1121
  
• 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 1121
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 298
  Illegal PstI site found at 1121
  
• 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 IMP-71 and T7 terminator. It encodes a protein which is IMP-71 fused with His-Sumo tag. The fusion protein is about 39.2 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

First, 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 PCR result of IMP-71. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

References

[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.