Part:BBa_K2933283

RBS+Linker h+His+Linker f+IMP-71+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+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 PstI site found at 852
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 93
  Illegal NheI site found at 877
  Illegal PstI site found at 852
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 852
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 852
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part is made up with three basic parts, the HiS tag, the thrombin restriction site and our target protein IMP-71. It encodes a protein which is IMP-71 fused with His tag. The fusion protein is about 27.2 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.

Figure 1. The PCR result of IMP-71.

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.