Part:BBa_K2933192
T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+JOHN-1+T7 terminator
The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+JOHN-1)and the biological module can be built into E.coil for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 390
Illegal XbaI site found at 47
Illegal PstI site found at 565 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 390
Illegal NheI site found at 167
Illegal NheI site found at 1244
Illegal PstI site found at 565 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 390
Illegal BglII site found at 279
Illegal BamHI site found at 478 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 390
Illegal XbaI site found at 47
Illegal PstI site found at 565 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 390
Illegal XbaI site found at 47
Illegal PstI site found at 565 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1089
Usage and Biology
This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of JOHN-1 and T7 terminator.It encodes a protein which is JOHN-1 fused with His and Sumo tag. The fusion protein is about 40.1 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of JOHN-1 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET28B-Sumo 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 JOHN-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.
References
[1]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).
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