[1]刘金泽,刘云惠,余子豪,等. 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[J].中国预防兽医学报,2018,(10):902-907.[doi:10.3969/j.issn.1008-0589.201712013]
 LIU Jin-ze,LIU Yun-hui,YU Zi-hao,et al. Establishment and performance analysis of a counter-selection cassette for Red recombination[J].Chinese journal of preventive veterinary medicine,2018,(10):902-907.[doi:10.3969/j.issn.1008-0589.201712013]
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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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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年10
页码:
902-907
栏目:
病原生物学
出版日期:
2018-11-15

文章信息/Info

Title:
 

Establishment and performance analysis of a counter-selection cassette for Red recombination

文章编号:
1008-0589(2018)10-0902-06
作者:
 

刘金泽刘云惠余子豪李永霞王明晓张远星李统战李倩文余旭平*

 

(浙江大学 动物科学学院,浙江 杭州 310058)


Author(s):
 

LIU Jin-ze LIU Yun-hui YU Zi-hao Li Yong-xia WANG Ming-xiao ZHANG Yuan-xing

LI Tong-zhan Li Qian-wen YU Xu-ping*

 

(College of Animal Science, Zhejiang University, Hangzhou 310058, China)



关键词:
 Red重组rpsL基因基因敲除大肠杆菌flgK基因
Keywords:
 

 Red recombination rpsL gene gene knock-out Escherichia coli flgK gene

分类号:
S852.61
DOI:
10.3969/j.issn.1008-0589.201712013
文献标志码:
A
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(strS,即rpsL基因)和卡那抗性基因(kanR),采用重叠延伸PCR方法将strS基因和kanR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PlacstrS-kanR打靶片段和ParastrS-kanR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8 000 μg/mL、2 000 μg/mL和500 μg/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。

  

Abstract:
 

To develop a method for deleting genes without leaving a selection marker in Escherichia coli, the strS-kanR 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 strS-kanR 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.

参考文献/References:

 

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(本文编辑:李 爽)

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更新日期/Last Update: 2018-11-28