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pBR322-Red在大肠杆菌lac操纵子基因敲除和敲入中的应用 被引量:1

Gene Knockout and Knockin on the Escherichia coli lac Operon Loci Using pBR322-Red System
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摘要 pBR32 2 - Red是一种新型重组工程系统 ,它携带了λ 噬菌体Red重组酶基因和一系列调控元件。对pBR32 2 Red最优重组条件进行探索后应用该质粒提供的体内同源重组功能 ,在菌株W3110体内 ,对染色体上的lac操纵子进行了基因修饰 ,包括 :①运用kan- sacB选择反选择方法和重叠引物方法敲除了阻遏基因lacI,②运用kan -sacB选择反选择方法和线性双链DNA介导的DNA重组方法将报告基因lacZ敲入lacA和lacY的位置 ,并且首次测定了报告基因lacZ在这三个结构基因位置的组成性表达情况。结果表明运用不同的重组策略 ,pBR32 2- Red系统都能方便有效地对大肠杆菌W3110染色体进行基因敲除和敲入修饰。 pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector,a series of regulatory elements of λ-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red,and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
出处 《生物工程学报》 CAS CSCD 北大核心 2005年第2期192-197,共6页 Chinese Journal of Biotechnology
基金 军队"十五"医药卫生科学基金资助 (No .0 1MA0 89)~~
关键词 体内 操纵子 基因敲除 BR 首次 携带 染色体 重组工程 报告基因 重组酶 pBR322-Red, homologous recombination, lac operon, knockout, knockin
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参考文献6

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共引文献4

同被引文献8

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  • 8周建光,洪鑫,黄翠芬.重组工程及其应用[J].Acta Genetica Sinica,2003,30(10):983-988. 被引量:16

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