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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===Usage and Biology===
 
===Usage and Biology===
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TMB-2 is a type of subclass B metal beta-lactamases, which is derived from TMB-1 mutation. The beta lactamases of the TMB family can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.<br>
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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
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==References==
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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>
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===References===
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[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;">
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   [[File:TMB-2-PCR.png|500px]]<br>
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   [[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


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.

TMB-2-PCR.png
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.