大肠埃希菌sdiA基因缺失株的建立
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摘要
目的为研究HSL信号分子对大肠埃希氏菌的影响而建立大肠埃希菌sdiA基因缺失株。方法通过Red重组系统敲除大肠杆菌BW25113和SM10λpir的sdiA基因,PCR测序鉴定;不同时间点检测A600值,绘制细菌生长曲线;荧光定量PCR检测csgD、csgB基因水平。结果 PCR测序结果经DNAman软件比对分析,与预想结果一致;与野生株相比,sdiA基因突变对细菌生长曲线无明显影响。结论成功建立BW25113、SM10λpir的sdiA基因缺失株,为后续研究铜绿假单胞菌信号分子HSL对大肠埃希菌的影响奠定了基础。
Objective To build sdiAgene muants of Escherichia coli BW25113 and SM10λpir strains.Methods The sdiAgene of Escherichia coli BW25113 and SM10λpir were knocked out with Red recombination system.The mutants were detected by PCR technique,and the following sequencing results were analyzed through alignment by software DNAman.Growth curve of strains were assayed by detecting the A600 value at different time point.The csgDand csgBgenes expression levels were detected by fluorescent quantitative PCR in SM10λpir sdiA mutant thereafter.Results PCR products electrophoresis showed a shorten fragment in mutants comparing to their parental strains BW25113 and SM10λpir,and it was confirmed by DNA sequencing.The growth curves and cells morphology in Gram stain were similar in both mutants and wild-type strains.The expression of csgDand csgBgenes was higher in SM10λpir sdiA mutants than those in wild-type strain.Conclusion The mutants of sdiA deletion of Escherichia coli BW25113 and SM10λpir were constructed successfully,and it will be the foundation for further research of the effects of the signal molecular HSL on Escherichia coli.
Objective To build sdiAgene muants of Escherichia coli BW25113 and SM10λpir strains.Methods The sdiAgene of Escherichia coli BW25113 and SM10λpir were knocked out with Red recombination system.The mutants were detected by PCR technique,and the following sequencing results were analyzed through alignment by software DNAman.Growth curve of strains were assayed by detecting the A600 value at different time point.The csgDand csgBgenes expression levels were detected by fluorescent quantitative PCR in SM10λpir sdiA mutant thereafter.Results PCR products electrophoresis showed a shorten fragment in mutants comparing to their parental strains BW25113 and SM10λpir,and it was confirmed by DNA sequencing.The growth curves and cells morphology in Gram stain were similar in both mutants and wild-type strains.The expression of csgDand csgBgenes was higher in SM10λpir sdiA mutants than those in wild-type strain.Conclusion The mutants of sdiA deletion of Escherichia coli BW25113 and SM10λpir were constructed successfully,and it will be the foundation for further research of the effects of the signal molecular HSL on Escherichia coli.
引文
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[2]Zhang L,Murphy PJ,Kerr A,et al.Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones[J].Nature,1993,362(6419):446-448.
[3]Fuqua C,Greenberg EP.Listening in on bacteria:acyl-homoserine lactone signalling[J].Nat Rev Mol Cell Biol,2002,3(9):685-695.
[4]Datsenko KA,Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products[J].Proc Natl Acad Sci U S A,2000,97(12):6640-6645.
[5]Zhou X,Meng X,Sun B.An EAL domain protein and cyclic AMP contribute to the interaction between the two quorum sensing systems in Escherichia coli[J].Cell Res,2008,18(9):937-948.
[6]Lee J,Maeda T,Hong SH,et al.Reconfiguring the quorum-sensing regulator sdiAof Escherichia coli to control biofilm formation via indole and N-acylhomoserine lactones[J].Appl Environ Microbiol,2009,75(6):1703-1716.
[7]García-Lara J,Shang LH,Rothfield LI.An extracellular factor regulates expression of sdiA,a transcriptional activator of cell division genes in Escherichia coli[J].J Bacteriol,1996,178(10):2742-2748.
[8]Wang XD,de Boer PA,Rothfield LI.A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli[J].EMBO J,1991,10(11):3363-3372.
[9]BendezúFO,de Boer PA.Conditional lethality,division defects,membrane involution,and endocytosis in mre and mrd shape mutants of Escherichia coli[J].J Bacteriol,2008,190(5):1792-1811.