Abstract
An improved and rapid genomic engineering method has been developed for the construction of custom-designed microorganisms by scarless chromosomal gene knockouts. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via scarless deletion of selected genomic regions. The deletion process is mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (1) an arabinose-inducible promoter that drives expression of λ-RED recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (2) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which accomplishes deletion of the introduced marker by double-strand breakage – mediated intramolecular recombination. This genomic deletion is performed simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette are nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hamilton C.M., Aldea M., Washburn B.K., Babitzke P. and Kushner S.R. (1989) New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol, 171, 4617–4622.
Link A.J., Phillips D. and Church G.M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol, 179, 6228–6237.
Posfai G., Kolisnychenko V., Bereczki Z. and Blattner F.R. (1999) Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res, 27, 4409–4415.
Murphy K.C. (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol, 180, 2063–2071.
Zhang Y., Buchholz F., Muyrers J.P. and Stewart A.F. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet, 20, 123–128.
Hashimoto M., Ichimura T., Mizoguchi H., Tanaka K., Fujimitsu K., Keyamura K., Ote T., Yamakawa T., Yamazaki Y., Mori H. et al. (2005) Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol Microbiol, 55, 137–149.
Kolisnychenko V., Plunkett G., 3rd, Herring C.D., Feher T., Posfai J., Blattner F.R. and Posfai G. (2002) Engineering a reduced Escherichia coli genome. Genome Res, 12, 640–647.
Posfai G., Plunkett G., 3rd, Feher T., Frisch D., Keil G.M., Umenhoffer K., Kolisnychenko V., Stahl B., Sharma S.S., de Arruda M. et al. (2006) Emergent properties of reduced-genome Escherichia coli. Science, 312, 1044–1046.
Copeland N.G., Jenkins N.A. and Court D.L. (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet, 2, 769–779.
Datsenko K.A. and Wanner B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A, 97, 6640–6645.
Lee E.C., Yu D., Martinez de Velasco J., Tessarollo L., Swing D.A., Court D.L., Jenkins N.A. and Copeland N.G. (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics, 73, 56–65.
Oppenheim A.B., Rattray A.J., Bubunenko M., Thomason L.C. and Court D.L. (2004) In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides. Virology, 319, 185–189.
Court D.L., Sawitzke J.A. and Thomason L.C. (2002) Genetic engineering using homologous recombination. Annu Rev Genet, 36, 361–388.
Yu B.J., Sung B.H., Koob M.D., Lee C.H., Lee J.H., Lee W.S., Kim M.S. and Kim S.C. (2002) Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol, 20, 1018–1023.
Choulika A., Perrin A., Dujon B. and Nicolas J.F. (1995) Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol, 15, 1968–1973.
Rong Y.S., Titen S.W., Xie H.B., Golic M.M., Bastiani M., Bandyopadhyay P., Olivera B.M., Brodsky M., Rubin G.M. and Golic K.G. (2002) Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev, 16, 1568–1581.
Schmidt-Puchta W., Orel N., Kyryk A. and Puchta H. (2004) Intrachromosomal homologous recombination in Arabidopsis thaliana. Methods Mol Biol, 262, 25–34.
Cox M.M., Layton S.L., Jiang T., Cole K., Hargis B.M., Berghman L.R., Bottje W.G. and Kwon Y.M. (2007) Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome. BMC Biotechnol, 7, 59.
Jamsai D., Orford M., Nefedov M., Fucharoen S., Williamson R. and Ioannou P.A. (2003) Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette. Genomics, 82, 68–77.
Kang Y., Durfee T., Glasner J.D., Qiu Y., Frisch D., Winterberg K.M. and Blattner F.R. (2004) Systematic mutagenesis of the Escherichia coli genome. J Bacteriol, 186, 4921–4930.
Sung B.H., Lee C.H., Yu B.J., Lee J.H., Lee J.Y., Kim M.S., Blattner F.R. and Kim S.C. (2006) Development of a biofilm production-deficient Escherichia coli strain as a host for biotechnological applications. Appl Environ Microbiol, 72, 3336–3342.
Tischer B.K., von Einem J., Kaufer B. and Osterrieder N. (2006) Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques, 40, 191–197.
Blattner F.R., Plunkett G., 3rd, Bloch C.A., Perna N.T., Burland V., Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew G.F. et al. (1997) The complete genome sequence of Escherichia coli K-12. Science, 277, 1453–1462.
Yu B.J., Kang K.H., Lee J.H., Sung B.H., Kim M.S. and Kim S.C. (2008) Rapid and efficient construction of markerless deletions in the Escherichia coli genome. Nucleic Acids Res, 36, e84.
Yu D., Ellis H.M., Lee E.C., Jenkins N.A., Copeland N.G. and Court D.L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A, 97, 5978–5983.
Acknowledgments
This work was supported in part by grants from 21C Frontier Program of Microbial Genomics and Applications (MG08-0204-1-0) from the Ministry of Education, Science and Technology and by grants from the Korea Science and Engineering Foundation (2008-0060733) and the Conversing Research Center Program through the National Research Foundation of Korea (2009-0082332).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sung, B.H., Lee, J.H., Kim, S.C. (2011). Scarless Chromosomal Gene Knockout Methods. In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_3
Download citation
DOI: https://doi.org/10.1007/978-1-61779-197-0_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-196-3
Online ISBN: 978-1-61779-197-0
eBook Packages: Springer Protocols