期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

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
下载PDF
导出
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.
作者 陈伟 于梅 李山虎 王鸣刚 周建光
机构地区 军事医学科学院生物工程研究所 厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室
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
分类号 Q78 [生物学—分子生物学] R378.21 [医药卫生—病原生物学]
  • 相关文献

参考文献6

  • 1Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet, 2001, 2(10): 769 - 779
  • 2Court DL, Sawitzke JA, Thomason LC. Genetic engineering using homologous recombination. Annu Rev Genet, 2002,36: 361 - 388
  • 3Yu D, Ellis HM, Lee EC et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA, 2000, 97(11) :5978 - 5983
  • 4Lee EC, Yu D, Martinez de Velasco J et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics,2001, 73(1):56-65
  • 5李山虎,洪鑫,于梅,陈伟,黄翠芬,周建光.Gap-Repair方式建立一种基于pBR322-Red的新型重组工程系统[J].Acta Genetica Sinica,2005,32(5):533-537. 被引量：5
  • 6Little JW. An exonuclease induced by bacteriophage lambda. Ⅱ .Nature of the enzymatic reaction. J Biol Chem, 1967, 242(4):679 - 686

二级参考文献9

  • 1Zhang Y M,Frank B,Muyrers J P,Francis S.A new logic for DNA engineering using recombination in Escherichia coli.Nature Genetics,1998,20:123～128.
  • 2Lee E C,Yu D G,de Martinez V J,Lino T,Deborah A S,Donald L C,Nancy A J,Neal G C.A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.Genomics,2001,73:56～65.
  • 3Zhang Y M,Muyrers J P,Giuseppe T,Francis S.DNA cloning by homologous recombination in Escherichia coli.Nature Biotectchnology,2000,18:1314～1317.
  • 4Muyrers J P,Zhang Y M,Giuseppe T,Francis S.Rapid modification of bacterial artificial chromosomes by ET-recombination.Nucleic Acids Research,1999,27:1555～1557.
  • 5Yu D G,Hilary M E,Lee E C,Nancy A J,Neal G C,Donald L C.An efficient recombination system for chromosome engineering in Escherichia coli.PNAS,2000,97:5978～5983.
  • 6Hilary M E,Yu D G,Tina D,Donald L C.High efficiency mutagenesis,repair,and engineering of chromosomal DNA using single-stranded oligonucleotides.PNAS,2001,98:6742～6746.
  • 7Kirill A D,Barry L W.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.PNAS,2000,97:6640～6645.
  • 8Donald L C,James A S,Lynn C.Thomason.Genetic engineering using homologous recombination.Annu Rev Genet,2002,36:361～388.
  • 9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

共引文献4

同被引文献8

  • 1Little JW.An exonuclease induced by bacteriophage lambda.Ⅱ.Nature of the enzymatic reaction.Journal of Biological Chemistry,1967,242(4):679-686.
  • 2Yu DG,Sawitzke JA,Ellis H,et al.Recombineering with overlapping single-stranded DNA oligonucleotides:testing a recombination intermediate.Proceedings of the National Academy of Sdences,2003,100(12):7207-7212.
  • 3Ellis HM,Yu D,DiTizio T,et al.High efficiency mutngenesis,repair,and engineering of chromosomal DNA using single-stranded oligonucleotides.Proceedings of the National Academy of Sciences,2001,98(12):6742-6746.
  • 4Yu D,Ellis HM,Lee EC,et al.An efficient recombination system for chromosome engineering in Escherichia coli.Proceedings of the National Academy of Sciences,2000,97(11):5978-5983.
  • 5Yin WX,Wu XS,Liu G,et al.Targeted correction of a chromosomal point mutation by modified single-stranded oligonucleotides in a GFP recovery system.Biochemical and Biophysical Research Conununications,2005,334:1032-1041.
  • 6Huen MSY,Lu LY,Liu DP.et al.Active transcription promotes single-stranded oligonucleotide mediated gene repair.Biochemical and Biophysical Research Communications,2007,353:33-39.
  • 7Sφren Warming,Nina Coatantino,Donald L Court,et al.Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Research,2005,33(4):e36.
  • 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

引证文献1

生物工程学报
2005年 第2期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部