Part:BBa_K2933210

T7 promoter+RBS b+Linker h+His+Linker f+CPS-1+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+CPS-1），and the biological module can be built into E.coli for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 47
  Illegal PstI site found at 741
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 169
  Illegal PstI site found at 741
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1023
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 47
  Illegal PstI site found at 741
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 47
  Illegal PstI site found at 741
  Illegal AgeI site found at 642
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein CPS-1. It encodes a protein which is CPS-1 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 33.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of CPS-1 . It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-28a 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 CPS-1.

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]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016