大肠杆菌基因组基因无痕敲除的优化方法

doi:10.13523/j.cb.20140610

中国生物工程杂志

2014, Vol. 34

Issue (06): 68-74 DOI: 10.13523/j.cb.20140610

技术与方法

大肠杆菌基因组基因无痕敲除的优化方法

葛高顺¹, 张立超¹, 赵昕², 胡学军¹, 李雅杰¹

1. 大连大学医学院大连 116622;
2. 辽宁出入境检验检疫局大连 116001

Optimization of the Method for Scarless Gene Knockout in Escherichia coli Genome

GE Gao-shun¹, ZHANG Li-chao¹, ZHAO Xin², HU Xue-jun¹, LI Ya-jie¹

1. Medical College, Dalian University, Dalian 116622, China;
2. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China

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摘要：

目的：优化大肠杆菌基因组基因无痕敲除的方法，提高无痕敲除的效率。方法：以无痕敲除大肠杆菌nanKETA基因簇为模型，利用Red同源重组系统和核酸内切酶I-SceI的筛选作用，通过两步连续同源重组无痕敲除大肠杆菌CLM37基因组中的nanKETA基因，优化无痕敲除时同源DNA长度与诱导用于筛选阳性克隆I-SceI表达的诱导剂浓度。通过比较敲除nanKETA基因前后菌株的生长曲线，研究大肠杆菌CLM37缺失nanKETA基因后的生长状态。结果：成功无痕敲除大肠杆菌CLM37基因组中的nanKETA基因，并在无痕化处理时，通过延长与基因组同源DNA的长度，由通常使用的80碱基对延长到684碱基对；并通过提高诱导筛选基因表达的四环素的浓度，由500 μg/ml提高到1000 μg/ml后，使无痕敲除效率高达90%以上。生长曲线研究表明，缺失nanKETA基因后的菌株生长状态与原菌株基本一致。结论：通过延长与基因组同源的双链核苷酸的长度和诱导筛选基因表达的四环素的浓度可显著提高无痕敲除的效率。

关键词： 大肠杆菌; 基因组; 无痕敲除; Red同源重组系统; I-SceI

Abstract:

Objective: The purpose was to improve the efficiency of scarless gene knockout in E. coli genome by the optimized method. Methods: The efficacy of scarless gene knockout in E.coli genome by two steps Red homologous recombinantion system and endonuclease I-SceI screening was investigated via optimization of the homologous DNA length with target sequence and the inducer concentration for production of I-SceI for the selection of positive clones. The scarless knockout of nanKETA clusters in the strain CLM37 was taken as a model. The new strain growth behaviour with the scarless knockout of nanKETA was investigated via the comparing the growth curves of wildtype E. coli CLM37. Results: The nanKETA clusters were knockouted scarless successfully in E. coli CLM37 genome, and the efficacy of scarless processing was increased up to 90% via extending the length of the homologous DNA with the genome, from the 80 base pairs normally used up to 684 base pairs, and increasing the concentration of the inducer tetracycline for producing I-SceI, from 500 μg/ml upto 1000 μg/ml. It is shown that the knockout of nanKETA clusters in E. coli CLM37 did not impact the growth. Conclusion: The efficacy of gene scarless knockout in E.coli can be significantly improved via extending the length of the homology DNA and increasing the concentration of inducer tetracycline.

Key words: Escherichia coli Genome Scarless gene knockout Red homologous recombination system I-SceI

收稿日期: 2014-03-05 出版日期: 2014-06-25

ZTFLH:

Q78

基金资助:

国家自然科学基金资助项目（31070822，31370937）

通讯作者: 胡学军, 李雅杰 E-mail: huxuejun@dlu.edu.cn;liyj@dlu.edu.cn

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引用本文:

葛高顺, 张立超, 赵昕, 胡学军, 李雅杰. 大肠杆菌基因组基因无痕敲除的优化方法[J]. 中国生物工程杂志, 2014, 34(06): 68-74.

GE Gao-shun, ZHANG Li-chao, ZHAO Xin, HU Xue-jun, LI Ya-jie. Optimization of the Method for Scarless Gene Knockout in Escherichia coli Genome. China Biotechnology, 2014, 34(06): 68-74.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140610 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I06/68

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