title: A method for generating precise gene deletions and insertions in Escherichia coli.
abstract: A simple and general method for disrupting chromosomal genes and introducing insertions is described. This procedure involves eliminating wild-type bacterial genes and introducing mutant alleles or other insertions at the original locus of the wild-type gene. To demonstrate the utility of this approach, the tig gene of Escherichia coli was replaced by homologous recombination with a cassette containing the chloramphenicol resistance gene and the sacB gene. The cassette was then removed and the tig mutant alleles were moved into the native tig location. Sequencing and Western blotting results demonstrated that insertions or deletions can be introduced precisely in E. coli using our approach. Our system does not require extra in vitro manipulations such as restriction digestion or ligation, and does not require use of specific plasmids or strains which are used to prevent false positive transformants caused by template plasmid transformation. This technique can be used widely in bacterial genome analysis.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, 100101, Beijing, China, zhqm@moon.ibp.ac.cn.
authors.forename: Qi-Ming
authors.initials: QM
authors.lastname: Zhou
authors.forename: Dong-Jie
authors.initials: DJ
authors.lastname: Fan
authors.forename: Jiang-Bi
authors.initials: JB
authors.lastname: Xie
authors.forename: Chuan-Peng
authors.initials: CP
authors.lastname: Liu
authors.forename: Jun-Mei
authors.initials: JM
authors.lastname: Zhou
references.citation: Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):15264-9
references.pmid: 11742086
references.citation: Nucleic Acids Res. 2001 Feb 1;29(3):E14
references.pmid: 11160916
references.citation: Gene. 2002 May 15;290(1-2):153-61
references.pmid: 12062810
references.citation: FEBS Lett. 1996 Apr 15;384(2):117-22
references.pmid: 8612805
references.citation: FEMS Microbiol Lett. 1999 Oct 1;179(1):153-60
references.pmid: 10481100
references.citation: J Microbiol Methods. 2008 Oct;75(2):359-61
references.pmid: 18619499
references.citation: J Bacteriol. 1997 Oct;179(20):6228-37
references.pmid: 9335267
references.citation: Nucleic Acids Res. 1999 Mar 1;27(5):1296-9
references.pmid: 9973617
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12017-22
references.pmid: 16091460
references.citation: Biochim Biophys Acta. 2008 Nov;1784(11):1728-34
references.pmid: 18539163
references.citation: Mol Syst Biol. 2006;2:2006.0008
references.pmid: 16738554
references.citation: Annu Rev Genet. 2002;36:361-88
references.pmid: 12429697
references.citation: Nucleic Acids Res. 2000 Jun 15;28(12):E65
references.pmid: 10871388
references.citation: Appl Environ Microbiol. 2008 Jul;74(13):4241-5
references.pmid: 18487404
references.citation: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
references.pmid: 10811905
references.citation: Genetics. 1997 Apr;145(4):877-89
references.pmid: 9093843
references.citation: Gene. 1993 May 15;127(1):15-21
references.pmid: 8486283
references.citation: Nat Biotechnol. 2000 Dec;18(12):1314-7
references.pmid: 11101815
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references.pmid: 10829079
references.citation: J Bacteriol. 1989 Sep;171(9):4617-22
references.pmid: 2548993
references.citation: J Mol Biol. 1997 Sep 5;271(5):827-37
references.pmid: 9299330
references.citation: Nucleic Acids Res. 1999 Mar 15;27(6):1555-7
references.pmid: 10037821
references.citation: Nucleic Acids Res. 1999 Nov 15;27(22):4409-15
references.pmid: 10536150
references.citation: Biochem Biophys Res Commun. 2004 Jan 16;313(3):509-15
references.pmid: 14697218
references.citation: Nat Genet. 1998 Oct;20(2):123-8
references.pmid: 9771703
references.citation: J Biol Chem. 2005 Oct 28;280(43):36079-87
references.pmid: 16141204
references.citation: Biochim Biophys Acta. 2009 Jun;1794(6):944-52
references.pmid: 19328871
references.citation: J Biol Chem. 2006 Oct 20;281(42):31963-71
references.pmid: 16926148
references.citation: J Biol Chem. 1997 Aug 29;272(35):21865-71
references.pmid: 9268318
references.citation: Nucleic Acids Res. 2003 Aug 1;31(15):e81
references.pmid: 12888533