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.
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Acknowledgments
We are indebted to Professor B. Wanner and the Coli Genetics Stock Center (CGSC) for the gift of plasmids and strains. This research was supported in part by the National Natural Science Foundation of China (30800548), a CAS Knowledge Innovation Grant (KSCX2-SW214-3) and the Ministry of Science and Technology of the People’s Republic of China (MOST, No.2007AA021405). The authors would like to thank Dr. Joy Fleming for her critical reading of this paper and helpful suggestions.
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Qi-Ming Zhou, Dong-Jie Fan have contributed equally to this work.
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Zhou, QM., Fan, DJ., Xie, JB. et al. A method for generating precise gene deletions and insertions in Escherichia coli . World J Microbiol Biotechnol 26, 1323–1329 (2010). https://doi.org/10.1007/s11274-009-0305-y
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DOI: https://doi.org/10.1007/s11274-009-0305-y