Part:BBa_K2933205
T7 promoter+RBS b+Linker h+His+Linker f+ElBlaII+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+ElBlA2-1），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
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal AgeI site found at 688 - 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 ElBla2-1. It encodes a protein which is ElBla2-1NDM-23 fused with His tag. The fusion protein is about 27.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of ElBla2-1 . 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 Elbla2-1. 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.
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
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