Difference between revisions of "Part:BBa K2933011"
 
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==Experimental results==
 
==Experimental results==
 
===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|]]        [[File:GIM-2-veri.jpeg|250px|]]<br>
 
   [[File:GIM-2-PCR.jpeg|400px|]]        [[File:GIM-2-veri.jpeg|250px|]]<br>
  
 
'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification
 
'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification
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===Exploration of expression condition===
 
  
  
  
 
==References==
 
==References==
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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.
+
[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.  
+
[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.
+
[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
+
[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
  
  

Latest revision as of 13:49, 24 September 2019


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