Difference between revisions of "Part:BBa K2933011"
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==Experimental results== | ==Experimental results== | ||
===Molecular cloning=== | ===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.<br> | ||
| + | |||
<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> | ||
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==References== | ==References== | ||
| − | 1 | + | [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 | + | [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 | + | [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 | + | [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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 731
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 731
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 731
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 731
- 1000COMPATIBLE 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.
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