Part:BBa_K2933018
subclass A metallo-beta-lactamase ARL-1, codon optimized in E. coli
This part encodes a protein called ARL-1, which is a beta-lactamase of subclass A.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 427
Illegal PstI site found at 541
Illegal PstI site found at 592 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 427
Illegal PstI site found at 541
Illegal PstI site found at 592 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 427
Illegal PstI site found at 541
Illegal PstI site found at 592 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 427
Illegal PstI site found at 541
Illegal PstI site found at 592 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
ARL-1 is a type of subclass A beta-lactamases, which is separated from Staphylococcus arlettae. The blaARL-1 was recently discovered and reported, that have a high mutation.
References
Molecular cloning
First, we used the vector pET-28a(+) and the vector pET28a-SUMO 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 ARL-1. 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.
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 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.
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