一种用于Red同源重组的正反筛选表达盒的构建及其反向筛选性能分析
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摘要
为建立能够应用于大肠杆菌基因无痕敲除的方法,本研究分别从大肠杆菌TG1基因组和pKD13质粒中扩增出链霉素敏感基因(str~S,即rpsL基因)和卡那抗性基因(kan~R),采用重叠延伸PCR方法将str~S基因和kan~R基因拼接,并将拼接片段分别克隆于pMD18-T和pBAD33载体中,再以重组载体为模板,以带有flgK基因同源臂的引物扩增获得Placstr~S-kan~R打靶片段和Parastr~S-kan~R打靶片段,然后结合Red重组技术构建了相应的flgK基因敲除菌株placSK-ΔflgK/TOP10和paraSK-ΔflgK/TOP10,并通过链霉素最小抑菌浓度(MIC)试验检测两种筛选系统的反向筛选性能。结果显示,野生型TOP10菌株、placSK-ΔflgK/TOP10菌株和paraSK-ΔflgK/TOP10菌株的链霉素MIC值分别为8 000μg/mL、2 000μg/mL和500μg/mL。表明采用两种筛选系统构建的敲除菌株均有一定的链霉素敏感表型,具有反向筛选的能力,但含Para启动子的筛选系统反向筛选性能更好。本研究建立了可用于大肠杆菌Red重组技术的反向筛选系统,为获得更有效的Red重组筛选工具提供了依据。
To develop a method for deleting genes without leaving a selection marker in Escherichia coli, the str~S-kan~R cassette with promoter lac or arabinose were constructed. And then the counter-selection cassette was electroporated into TOP10 strain after flanked by homology arms of flgK gene to produce recombinant strains of TOP10_PlacSK_ΔflgK and TOP10_ParaSK_ΔflgK.Moreover, the minimal inhibitory concentration(MIC) assay was performed to evaluate the sensitivity of streptomycin for recombinant strains. The results showed that the MIC of wild type TOP10 strain, TOP10_PlacSK_ΔflgK strain and TOP10_ParaSK_ΔflgK strain were 8,000μg/mL, 2,000μg/mL and 500μg/mL respectively, which indicated that both recombinant strains were sensitive to streptomycin, and the counter-selection efficiency of the str~S-kan~R cassette with promoter arabinose was much better. This study established a counter-selection system for deleting genes in E.coli and provided foundations for optimization of Red recombination.
To develop a method for deleting genes without leaving a selection marker in Escherichia coli, the str~S-kan~R cassette with promoter lac or arabinose were constructed. And then the counter-selection cassette was electroporated into TOP10 strain after flanked by homology arms of flgK gene to produce recombinant strains of TOP10_PlacSK_ΔflgK and TOP10_ParaSK_ΔflgK.Moreover, the minimal inhibitory concentration(MIC) assay was performed to evaluate the sensitivity of streptomycin for recombinant strains. The results showed that the MIC of wild type TOP10 strain, TOP10_PlacSK_ΔflgK strain and TOP10_ParaSK_ΔflgK strain were 8,000μg/mL, 2,000μg/mL and 500μg/mL respectively, which indicated that both recombinant strains were sensitive to streptomycin, and the counter-selection efficiency of the str~S-kan~R cassette with promoter arabinose was much better. This study established a counter-selection system for deleting genes in E.coli and provided foundations for optimization of Red recombination.
引文
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[9]CLSI M100-S19. Performance standards for antimicrobial susceptibility testing[S].
[10]姜露,聂佳佳,杨样,等. K88ac+和K88ad+产肠毒素大肠杆菌hlyA基因缺失株的构建及相关功能初步分析[J].中国预防兽医学报,2014,36(07):524-529.
[11]黄博言,祁克宗,涂健,等.禽致病性大肠杆菌pho P/phoQ基因敲除株的构建及其毒力基因转录调控的研究[J].中国预防兽医学报,2015,37(07):518-523.
[12]Zhou Qi-ming, Fan Dong-jie, Xie Jiang-bi, et al. A method for generating precise gene deletions and insertions in Escherichia coli[J]. World J Microbiol Biotechnol, 2010, 26(7):1323-1329.
[13]Zhang Xue-li, Jantama K, Moore J C, et al. Production of L-alanine by metabolically engineered Escherichia coli[J]. Appl Microbiol Biotechnol, 2007, 77(2):355-366.
[14]Lee D J, Bingle L E, Heurlier K, et al. Gene doctoring:A method for recombineering in laboratory and pathogenic Escherichia coli strains[J]. BMC Microbiol, 2009, 9(12):252-266.
[15]Kinoshita M. Interactions of bacterial flagellar chaperone-substrate complexes with FlhA contribute to co-ordinating assembly of the flagellar filament[J]. Mol Microbiol, 2013, 90(6),1249-1261.
[2]Datta S, Costantino N, Court D L. A set of recombineering plasmids for gramnegative bacteria[J]. Gene, 2006, 379:109-115.
[3]胡逢雪,丁锐,崔震海,等.大肠杆菌基因无痕敲除技术及策略[J].生物技术通讯,2013,24(04):552-557.
[4]Kaczmarczyk A, Vorholt J A, Francez-Charlot A. Markerless gene deletion system for Sphingomonads[J]. Appl Environ Microbiol, 2012, 78(10):3774.
[5]Kim I K, Jeong W K, Lim S H, et al. The small ribosomal protein S12P gene rpsL as an efficient positive selection marker in allelic exchange mutation systems for Corynebacterium glutamicum[J]. J Microbiol Methods, 2011, 84(1):128-130.
[6]Reyrat J M, Pelicic V, Gicquel B, et al. Counterselectable markers:Untapped tools for bacterial genetics and pathogenesis[J].Infect Immun, 1998, 66(9):4011-4017.
[7]Wang Shu-wen, Zhao Yuan-jun, Leiby M, et al. A new positive/negative selection scheme for precise BAC recombineering[J]. Mol Biotechnol, 2009, 42(1):110-116.
[8]张雪,温廷益. Red重组系统用于大肠杆菌基因修饰研究进展[J].中国生物工程杂志,2008,28(12):89-93.
[9]CLSI M100-S19. Performance standards for antimicrobial susceptibility testing[S].
[10]姜露,聂佳佳,杨样,等. K88ac+和K88ad+产肠毒素大肠杆菌hlyA基因缺失株的构建及相关功能初步分析[J].中国预防兽医学报,2014,36(07):524-529.
[11]黄博言,祁克宗,涂健,等.禽致病性大肠杆菌pho P/phoQ基因敲除株的构建及其毒力基因转录调控的研究[J].中国预防兽医学报,2015,37(07):518-523.
[12]Zhou Qi-ming, Fan Dong-jie, Xie Jiang-bi, et al. A method for generating precise gene deletions and insertions in Escherichia coli[J]. World J Microbiol Biotechnol, 2010, 26(7):1323-1329.
[13]Zhang Xue-li, Jantama K, Moore J C, et al. Production of L-alanine by metabolically engineered Escherichia coli[J]. Appl Microbiol Biotechnol, 2007, 77(2):355-366.
[14]Lee D J, Bingle L E, Heurlier K, et al. Gene doctoring:A method for recombineering in laboratory and pathogenic Escherichia coli strains[J]. BMC Microbiol, 2009, 9(12):252-266.
[15]Kinoshita M. Interactions of bacterial flagellar chaperone-substrate complexes with FlhA contribute to co-ordinating assembly of the flagellar filament[J]. Mol Microbiol, 2013, 90(6),1249-1261.