Difference between revisions of "Part:BBa K2933170"
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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with three basic parts(Tac promoter，RBS a and Linker g)and a composite part(GST+Linker e+GIM-2).It encodes a protein which is GIM-2 fused with GST tag. The fusion protein is about 53.4 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of GIM-2 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
  
 
===Molecular cloning===
 
===Molecular cloning===

Revision as of 14:20, 21 September 2019


Tac promoter+RBS a+Linker g+GST+Linker e+GIM-2

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+GIM-2),and the biological module can be built into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1525
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1525
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1525
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1525
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 181


Usage and Biology

This composite part is made up with three basic parts(Tac promoter，RBS a and Linker g)and a composite part(GST+Linker e+GIM-2).It encodes a protein which is GIM-2 fused with GST tag. The fusion protein is about 53.4 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of GIM-2 and combine the two parts with the cutting site of Prescission Protease. 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

GIM-2-PCR.jpeg GIM-2-veri.jpeg
Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification

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