Coding

Part:BBa_K2933011

Designed by: Ruihan Dong   Group: iGEM19_TJUSLS_China   (2019-09-06)


subclass B1 metallo-beta-lactamase GIM-2, codon optimized in E. coli

This part encodes a protein called GIM-2, which is a metallo-beta-lactamase of subclass B1.


Usage and Biology

GIM-2 is a new variant of GIM-1 with a single mutation, A290G, which was recently discovered. The German imipenemase-1 (GIM-1) MBL was first identified in clinical isolates of Pseudomonas aeruginosa in Germany in 2002. Recently, GIM-1 has been identified in other bacterial species, such as Serratia marcescens, Enterobacter cloacae, and Acinetobacter pittii , indicating that it is transmitted on mobile genetic elements. As a typical type of metallo-beta-lactamases which make bacteria antibiotic-resistant, it can hydrolyze extensive substrate and may pose a threat to human life and health.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 731
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 731
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 731
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 731
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental results

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 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


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