Difference between revisions of "Part:BBa K2933191"
 
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===Molecular cloning===
 
===Molecular cloning===
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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.<br>
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<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:GIM-2-PCR.jpeg|400px|]]<br>
 
   [[File:GIM-2-PCR.jpeg|400px|]]<br>

Latest revision as of 02:03, 26 September 2019


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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 1220
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 390
    Illegal BglII site found at 279
    Illegal BamHI site found at 478
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 1220
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 1220
  • 1000
    COMPATIBLE 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.

GIM-2-PCR.jpeg
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