Part:BBa_K2933222
T7 promoter+RBS b+Linker h+His+Linker f+Fla.103+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+Fla.103），and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 47
Illegal PstI site found at 280 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 280 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 762
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 280 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 280 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology=
This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein Fla.103. It encodes a protein which is Fla.103 fused with His tag. The fusion protein is about 28.7 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Fla.103. 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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