Difference between revisions of "Part:BBa K2933001"
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===Expression and purification=== | ===Expression and purification=== | ||
Pre-expression:<br> | Pre-expression:<br> | ||
− | The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ overnight | + | The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br> |
Massive expressing:<br> | Massive expressing:<br> | ||
− | After | + | After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours).<br> |
Purification of GST fusion proteins:<br> | Purification of GST fusion proteins:<br> |
Revision as of 13:43, 3 September 2019
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
Molecular cloning
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours).
Purification of GST fusion proteins:
Anion exchange column:
Gel filtration chromatography: