Part:BBa_K2933262
RBS b+Linker h+His+Linker a+Sumo+Linker b+IMP-71+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+IMP-71) and T7 terminator,and 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 EcoRI site found at 298
Illegal PstI site found at 1121 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 298
Illegal NheI site found at 75
Illegal NheI site found at 1146
Illegal PstI site found at 1121 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 298
Illegal BglII site found at 187
Illegal BamHI site found at 386 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 298
Illegal PstI site found at 1121 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 298
Illegal PstI site found at 1121 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Bacteria with IMP-type enzymes have spread through out the world, and the IMP group now has more than 50 variants
These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin
References
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 IMP-71. 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.
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