Abstract
Lambda Red recombineering is an easy and efficient method for generating genetic modifications in Escherichia coli. For gene deletions, lambda Red recombineering is combined with the use of selectable markers, which are removed through the action of, e.g., flippase (Flp) recombinase. This PCR-based engineering method has also been applied to a number of other bacteria. In this chapter, we describe a recently developed one plasmid-based method as well as the use of a strain with genomically integrated recombineering genes, which significantly speeds up the engineering of strains with multiple genomic alterations.
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References
Karu AE, Sakaki Y, Echols H, Linn S (1975) The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli. J Biol Chem 250(18):7377–7387
Murphy KC (1991) Lambda gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. J Bacteriol 173(18):5808–5821
Kulkarni SK, Stahl FW (1989) Interaction between the sbcC gene of Escherichia coli and the gam gene of phage lambda. Genetics 123(2):249–253
Subramanian K, Rutvisuttinunt W, Scott W, Myers RS (2003) The enzymatic basis of processivity in lambda exonuclease. Nucleic Acids Res 31(6):1585–1596
Little JW (1967) An exonuclease induced by bacteriophage lambda. II Nature of the enzymatic reaction. J Biol Chem 242(4):679–686
Karakousis G, Ye N, Li Z, Chiu SK, Reddy G, Radding CM (1998) The beta protein of phage lambda binds preferentially to an intermediate in DNA renaturation. J Mol Biol 276(4):721–731. doi:10.1006/jmbi.1997.1573
Kmiec E, Holloman WK (1981) Beta protein of bacteriophage lambda promotes renaturation of DNA. J Biol Chem 256(24):12636–12639
Radding CM, Rosensweig J, Richards F, Cassuto E (1971) Separation and characterization of exonuclease B protein, and a complex of both. J Biol Chem 246:2510–2512
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97(11):5978–5983. doi:10.1073/pnas.100127597
Yu D, Sawitzke JA, Ellis H, Court DL (2003) Recombineering with overlapping single-stranded DNA oligonucleotides: testing a recombination intermediate. Proc Natl Acad Sci U S A 100(12):7207–7212. doi:10.1073/pnas.1232375100
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4(2):206–223. doi:10.1038/nprot.2008.227
Maresca M, Erler A, Fu J, Friedrich A, Zhang Y, Stewart AF (2010) Single-stranded heteroduplex intermediates in lambda Red homologous recombination. BMC Mol Biol 11:54. doi:10.1186/1471-2199-11-54
Mosberg JA, Lajoie MJ, Church GM (2010) Lambda Red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186(3):791–799. doi:10.1534/genetics.110.120782
Jensen SI, Lennen RM, Herrgard MJ, Nielsen AT (2015) Seven gene deletions in seven days: fast generation of Escherichia coli strains tolerant to acetate and osmotic stress. Sci Rep 5:17874. doi:10.1038/srep17874
Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158(1):9–14
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12):6640–6645. doi:10.1073/pnas.120163297
Cox MM (1983) The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. Proc Natl Acad Sci U S A 80(14):4223–4227
Gronostajski RM, Sadowski PD (1985) The FLP protein of the 2-micron plasmid of yeast. Inter- and intramolecular reactions. J Biol Chem 260(22):12328–12335
Senecoff JF, Bruckner RC, Cox MM (1985) The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc Natl Acad Sci U S A 82(21):7270–7274
Yamamoto N, Nakahigashi K, Nakamichi T, Yoshino M, Takai Y, Touda Y, Furubayashi A, Kinjyo S, Dose H, Hasegawa M, Datsenko KA, Nakayashiki T, Tomita M, Wanner BL, Mori H (2009) Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 5:335. doi:10.1038/msb.2009.92
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(2006):0008. doi:10.1038/msb4100050
Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28(1):27–30
Zhou J, Rudd KE (2013) EcoGene 3.0. Nucleic Acids Res 41(Database issue):D613–D624. doi:10.1093/nar/gks1235
Acknowledgments
The development of the methods described in this chapter was obtained through funding from the Novo Nordisk Foundation. pKD3, pKD4, pSIJ8, and strain SIJ488 are all available from Addgene. Other plasmids are available upon request from the authors.
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Jensen, S.I., Nielsen, A.T. (2018). Multiplex Genome Editing in Escherichia coli . In: Jensen, M.K., Keasling, J.D. (eds) Synthetic Metabolic Pathways. Methods in Molecular Biology, vol 1671. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7295-1_8
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DOI: https://doi.org/10.1007/978-1-4939-7295-1_8
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