Difference between revisions of "Part:BBa K2933253"
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===Usage and Biology===
 
===Usage and Biology===
  
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GIM-2 is a new variant of GIM-1 with a single mutation, A290G, which was recently discovered.
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein GIM-2 and T7 terminator. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
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The German imipenemase-1 (GIM-1) MBL was first identified in clinical isolates of Pseudomonas aeruginosa in Germany in 2002.
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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.  
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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.
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Revision as of 13:19, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+GIM-2+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+GIM-2) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 1128
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1158
    Illegal PstI site found at 1128
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 1128
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 1128
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein GIM-2 and T7 terminator. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.


Experimental results

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