Part:BBa_K2933265
RBS+Linker h+His+Linker f+NDM-23+T7 terminator
This part consists of RBS, Linker h, protein coding sequence(His+Linker f+NDM-23) 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 93
Illegal NheI site found at 949 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with six basic parts(RBS , Linker h, His, Linker f , NDM-23 and T7 terminator ). It encodes a protein which is NDM-23 fused with His tag. The fusion protein is about 28.5 kD. It is convenient for us to purify our target protein.
Molecular cloning
We used the vector pET28a 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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