Part:BBa_K2933001
subclass B1 metallo-beta-lactamase NDM-23, codon optimized in E. coli
This part encodes a kind of beta lactamase called NDM-23.
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
NDM-23 is a type of subclass B metal beta-lactamases, which is derived from NDM-1 mutation. The beta lactamases of the NDM 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
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with ampicillin.
Massive expressing:
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h.
Purification of GST fusion proteins:
Anion exchange column:
Gel filtration chromatography:
Enzyme activity determination
Determination of enzyme concentration
Determination of the buffer composition
Determination of substrate concentration
Determination of the mikkelsen curve
Hydrolysis efficiency of different antibiotics
Inhibition of different inhibitors
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