Part:BBa_K2933221
T7 promoter+RBS b+Linker h+His+Linker f+IMP-71+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+IMP-71），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 928 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 928 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 928 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 928 - 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 terminatorand and our target protein IMP-71. It encodes a protein which is IMP-71 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 27.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of IMP-71 . 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 IMP-71.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
References
[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.
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