Difference between revisions of "Part:BBa K2933015"
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This part encodes a protein called TMB-2, which is a metallo-beta-lactamase of subclass B1. | This part encodes a protein called TMB-2, which is a metallo-beta-lactamase of subclass B1. | ||
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The Tripoli metallo-beta-lactamase-1 (TMB-1) gene was first discovered in a Achromobacter xylosoxidans strain obtained from an environmental sample in a hospital in Tripoli, Libya, in 2011 | The Tripoli metallo-beta-lactamase-1 (TMB-1) gene was first discovered in a Achromobacter xylosoxidans strain obtained from an environmental sample in a hospital in Tripoli, Libya, in 2011 | ||
After the initial report, TMB-1 has been identified in clinical isolates of Acinetobacter spp. in Japan, and the new TMB-1 variant named TMB-2, with the single mutation S228P, was isolated from a different hospital in Japan also in clinical isolates of Acinetobacter spp.<br> | After the initial report, TMB-1 has been identified in clinical isolates of Acinetobacter spp. in Japan, and the new TMB-1 variant named TMB-2, with the single mutation S228P, was isolated from a different hospital in Japan also in clinical isolates of Acinetobacter spp.<br> | ||
| − | ==References== | + | ===References=== |
| − | + | [1]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8). | |
===Molecular cloning=== | ===Molecular cloning=== | ||
First, we used the vector pGEX-6p-1 and pET-28b_SUMO to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br> | First, we used the vector pGEX-6p-1 and pET-28b_SUMO 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:TMB-2-PCR.png| | + | [[File:TMB-2-PCR.png|400px]]<br> |
'''Figure 1.''' Left: The PCR result of TMB-2. Right: The verification results by enzyme digestion.<br> | '''Figure 1.''' Left: The PCR result of TMB-2. Right: The verification results by enzyme digestion.<br> | ||
</p> | </p> | ||
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br> | After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br> | ||
Latest revision as of 14:55, 23 September 2019
subclass B1 metallo-beta-lactamase TMB-2, codon optimized in E. coli
This part encodes a protein called TMB-2, which is a metallo-beta-lactamase of subclass B1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The Tripoli metallo-beta-lactamase-1 (TMB-1) gene was first discovered in a Achromobacter xylosoxidans strain obtained from an environmental sample in a hospital in Tripoli, Libya, in 2011
After the initial report, TMB-1 has been identified in clinical isolates of Acinetobacter spp. in Japan, and the new TMB-1 variant named TMB-2, with the single mutation S228P, was isolated from a different hospital in Japan also in clinical isolates of Acinetobacter spp.
References
[1]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).
Molecular cloning
First, we used the vector pGEX-6p-1 and pET-28b_SUMO 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 PCR result of TMB-2. Right: The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.