Part:BBa_K2933168

Tac promoter+RBS a+Linker g+GST+Linker e+CPS-1

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+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 PstI site found at 1331
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1331
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1613
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1331
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1331
  Illegal AgeI site found at 1232
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 181
  

Usage and Biology

This composite part is made up with three basic parts(Tac promoter，RBS a and Linker g)and a composite part(GST+Linker e+CPS-1). It encodes a protein which is CPS-1 fused with GST tag. The fusion protein is about 59.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of CPS-1 and combine the two parts with the cutting site of Prescission Protease. 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

First, we used the vector pGEX-6p-1 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 CPS-1. 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]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