Part:BBa_K2933015

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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.