Part:BBa_K2933191
T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+GIM-2+T7 terminator
The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+GIM-2)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 1220 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 390
Illegal NheI site found at 167
Illegal NheI site found at 1250
Illegal PstI site found at 1220 - 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 1220 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 390
Illegal XbaI site found at 47
Illegal PstI site found at 1220 - 1000COMPATIBLE WITH RFC[1000]
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 GIM-2 and T7 terminator.It encodes a protein which is GIM-2 fused with His and Sumo tag. The fusion protein is about 39.4 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of GIM-2 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 pGEX-6p to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. The result of PCR
References
[1]Skagseth S , Akhter S , Paulsen M H , et al. Metallo-β-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding[J]. European Journal of Medicinal Chemistry, 2017, 135:159-173.
[2]Wendel AF, MacKenzie CR. 2015. Characterization of a novel metallo-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany. Antimicrob Agents Chemother 59:1824 –1825.
[3]Borra P S , Samuelsen O , Spencer J , et al. Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-beta-Lactamases[J]. Antimicrobial Agents and Chemotherapy, 2013, 57(2):848-854.
[4]Susann S, Trine J C, Gro Elin K B, James S, Ørjan S, Hanna-Kirsti S. L. Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1. Antimicrobial Agents and Chemotherapy Jan 2016, 60 (2) 990-1002
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