Part:BBa_K2933260
RBS b+Linker h+His+Linker a+Sumo+Linker b+ARL-1+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ARL-1) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 298
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 298
Illegal NheI site found at 75
Illegal NheI site found at 1254
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 298
Illegal BglII site found at 187
Illegal BamHI site found at 386 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 298
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 298
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 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.
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.
None |