Part:BBa_K2933242
RBS a+Linker g+GST+Linker e+Fla.103
This part consists of RBS a, protein coding sequence(GST+Linker e+Fla.103). The blaFla.103 is separated from Flavobacterium sp. 103. 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
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1284
- 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
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 Fla.103. It encodes a protein which is Fla.103 fused with GST tag. The fusion protein is about 54.7 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Fla.103 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.We then extracted plasmids and performed double enzyme digestion verification.
Figure 1. (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the
results verified by double 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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