Part:BBa_K2933233
RBS a+Linker g+GST+Linker e+JOHN-1
This part consists of RBS a, protein coding sequence(GST+Linker e+JOHN-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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 802
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 802
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 802
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 802
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 113
Illegal SapI.rc site found at 1326
Usage and Biology
This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein 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.
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).
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.
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