Coding

Part:BBa_K2933015

Designed by: Yiwen Ye   Group: iGEM19_TJUSLS_China   (2019-09-08)
Revision as of 08:48, 8 September 2019 by Yiwenye (Talk | contribs)


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

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.

References

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.

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