Difference between revisions of "Part:BBa K2933013"
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===Molecular cloning=== | ===Molecular cloning=== | ||
Revision as of 08:07, 8 September 2019
subclass B1 metallo-beta-lactamase SHD, codon optimized in E. coli
This part encodes a protein called VIM-66, which is a metallo-beta-lactamase of subclass B1.
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
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
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: