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 seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminator 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-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. The PCR result of Elbla2-1.
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.
References
[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]
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