Difference between revisions of "Part:BBa K2933013"
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===Usage and Biology=== | ===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.<br> | |
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===Molecular cloning=== | ===Molecular cloning=== | ||
| − | + | First, we used the vector pGEX-6p-1 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;"> | ||
| + | [[File:SHD-PCR.png|500px]]<br> | ||
| + | '''Figure 1.''' Left: The PCR result of SHD. Right: The verification results by enzyme digestion.<br> | ||
| + | </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> | ||
===Exploration of expression condition=== | ===Exploration of expression condition=== | ||
| − | [[File:SHD- | + | [[File:SHD-6p.jpeg|160px|left|]] |
Revision as of 07:38, 15 September 2019
subclass B1 metallo-beta-lactamase SHD, codon optimized in E. coli
This part encodes a protein called SHD, which is a metallo-beta-lactamase of subclass B1.
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.
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]
Molecular cloning
First, we used the vector pGEX-6p-1 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 SHD. 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.
Exploration of expression condition
Figure 2.
The result of SDS-PAGE.
Lane1, uninduced SHD-28a(29.0kD).
Lane2, 37°C induced SHD-28a(29.0kD)with 0.5mM IPTG.
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with kanamycin(50 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
After taking samples, we transfer them into 1L LB medium and add antibiotic to 50 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). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 rpm for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.
Purification of GST fusion proteins: