Part:BBa_K2933171
Tac promoter+RBS a+Linker g+GST+Linker e+JOHN-1
This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+JOHN-1),and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 870
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 870
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 870
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 870
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 181
Illegal SapI.rc site found at 1394
Usage and Biology
This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and JOHN-1). It encodes a protein which is JOHN-1 fused with GST tag. The fusion protein is about 54.1 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of JOHN-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 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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