Part:BBa_K2933230

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

This part consists of RBS a, protein coding sequence(GST+Linker e+CPS-1), the RBS and the protein coding sequence can be connected by linker g. 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 1263
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1263
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1545
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1263
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1263
  Illegal AgeI site found at 1164
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 113
  

Usage and Biology

blaCPS-1, a new carbapenem-hydrolyzing beta-lactamases, this enzyme has not yet emerged in clinical settings but constitute potential carbap- enem resistance determinants in pathogenic bacterial species, as demonstrated by their ability to confer resistance to ampi- cillin and various cephalosporins, as well as reduced suscepti- bility to carbapenems, once expressed in E. coli.

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