Part:BBa_K2933182
T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+NDM-23+T7 terminator
The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+NDM-23)and the biological module can be built into E.coil for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 390
Illegal XbaI site found at 47 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 390
Illegal NheI site found at 167
Illegal NheI site found at 1310 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 390
Illegal BglII site found at 279
Illegal BamHI site found at 478 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 390
Illegal XbaI site found at 47 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 390
Illegal XbaI site found at 47 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of NDM23 and T7 terminator.It encodes a protein which is NDM-23 fused with His and Sumo tag. The fusion protein is about 54.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of NDM-23 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.
Molecular cloning
We used the vector pET28B-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 NDM-23. 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.
References
[1] Van Duin D, Doi Y. The global epidemiology of carbapenemase-producing Enterobacteriaceae [J]. Virulence, 2017,8(4): 460469.
[2] Yong D, Toleman MA, Giske cG, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique geneticstructure in Klebsiella pneumoniae sequence type 14 from India [J]. Antimicrob Agents Chemother, 2009,53(12): 5046-5054.
[3] Wu w. Feng Y, Tang G et al. NDM Metallo-ß-Lactamases and Their Bacterial Producers in Health Care Sttings [J]. Clin Microbiol Rev, 2019,32(2): 0011500118.
[4] Khan AU, Maryam L, Zarilli R. Structure, Genetics and Worldwide Spread of New Delhi Maeallo-beta-lactamase (NDM): a threat to public health [J].BMC Microbiol, 2017,17(1):101-112.
[5] Zheng B, Lv T, Xu H, et al. Discovery and characterisation of an escherichia coli ST206 strain producing NDM-5 and MCR-1 from a patient with acute diarrhoea in China [J]. Int JAntimicrob Agents, 2018,51(2): 273-275.
[6] Li X, Jiang Y, Wu K,et al. Whole-genome sequencing identification of a multidrug-resistan t Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China [J]. Infect Genet Evol, 2017,55: 195-198.
[7] Rahman M, Shukla SK, Prasad KN, et al. Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-I, NDM-5, NDM-6 and NDM-7 in multidrug- resistant Enterobacteriaceae from India [J]. Int J Antimierob Agents, 2014，44(1).
[8] Rojas LJ, Hujer AM, Rudin SD, et al. NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas [J]. Antimicrob Agents Chemother,2017,61(7): pii: e00454-17.
[9] Almakki A, Maure A, Pantel A, et al. NDM-5-producing Escherichia coli in an urban river in Montpellier, France [I]. Int J Antimicrob Agents, 2017,50(1): 123-124.
[10] Rozales FP, Magagnin cM, Campos JC, et al. Characterization of Transformants Obtained From NDM-1-Producing Enterobacteriaceae in Brazil [J]. Infect Control Hosp Epidemiol,2017,38(5): 634-636.
[11] Yang B, Feng Y, McNally A, et al. Occurrence of Enterobacter hormaechei carrying blaNDM-1 and blaKPC-2 in China [J]. Diagn Microbiol Infect Dis, 2018.90(2): 139-142.
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