Part:BBa_K2933263
RBS b+Linker h+His+Linker a+Sumo+Linker b+Fla.103+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+Fla.103) 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 473 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 298
Illegal NheI site found at 75
Illegal NheI site found at 1158
Illegal PstI site found at 473 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 298
Illegal BglII site found at 187
Illegal BglII site found at 955
Illegal BamHI site found at 386 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 298
Illegal PstI site found at 473 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 298
Illegal PstI site found at 473 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Fla.103 is a type of subclass B metal beta-lactamases. The beta lactamases can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.
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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