Difference between revisions of "Part:BBa K2933014"
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===Molecular cloning=== | ===Molecular cloning=== | ||
| − | First,we obtained BcII by PCR. | + | First,we obtained BcII by PCR.<br> |
[[File:BcII-194 PCR.jpeg|200px|]]<br> | [[File:BcII-194 PCR.jpeg|200px|]]<br> | ||
'''Figure 1.''' The PCR result of BcII.<br> | '''Figure 1.''' The PCR result of BcII.<br> | ||
| − | + | ||
Then 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.After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br> | Then 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.After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br> | ||
Revision as of 08:35, 8 September 2019
BCII family subclass B1 metallo-beta-lactamase, codon optimized in E. coli
This part encodes a protein called BcII, 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
BcII is a type of subclass B metallo-beta-lactamase found in Bacillus anthracis. It can hydrolyzes Beta-lactam antibiotics to make pathogenic bacteria produce drug resistance. The bacterium Bacillus anthracis is responsible for causing anthrax infection, which is often fatal. Because symptoms arising from the bacterial infection are similar to a common cold,misdiagnosis in the early stage is possible and frequent. So the clinical strains carrying it become a great threat to human life and health. Beta-Lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are commercially available and can be used with existing antibiotics to cure some antibiotic resistant infections, but these are not effective against MBLs. Although some potential inhibitors have the ability to inhibit it, there are no commercially available inhibitors of it. We use the protein to carry out high-throughput screening to find inhibitors that can inhibit it.
References
Sung-Kun Kim, Mara Demuth, Sara R. Schlesinger, Sung Joon Kim, Jonathan Urbanczyk, Robert W. Shaw & Hyunshun Shin (2016) Inhibition of Bacillusanthracis metallo-β- lactamase by compounds with hydroxamic acid functionality, Journal of Enzyme Inhibition and Medicinal Chemistry, 31:sup4, 132-137, DOI: 10.1080/14756366.2016.1222580
Molecular cloning
First,we obtained BcII by PCR.
Figure 1. The PCR result of BcII.
Then 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.After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
Figure 2. The verification results by enzyme digestion.
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 transfered 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). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.