Part:BBa_K2933275
RBS+Linker h+His+Linker f+JOHN-1+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+JOHN-1) 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 204
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 93
Illegal NheI site found at 883
Illegal PstI site found at 204 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 204
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 204
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 728
Usage and Biology
This composite part is made up with four basic parts(Tac promoter，RBS a , Linker g , GST+Linker e+JOHN-1). It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 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. Left: The PCR result of JOHN-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]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).
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