Abstract
Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligonucleotide recombination is one type of recombineering that uses ssDNA oligonucleotides to direct chromosomal mutations. Oligo recombination occurs without addition of any exogenous functions, making this approach potentially useful in many different bacteria. Here we describe the basic technique for constructing a site-specific genomic mutation in Pseudomonas syringae.
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References
Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071
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 97:5978–5983
Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128
van Kessel JC, Marinelli LJ, Hatfull GF (2008) Recombineering mycobacteria and their phages. Nat Rev Microbiol 6:851–857
Datta S, Costantino N, Zhou X, Court DL (2008) Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proc Natl Acad Sci U S A 105:1626–1631
Swingle B, Markel E, Costantino N, Bubunenko MG, Cartinhour S, Court DL (2010) Oligonucleotide recombination in Gram-negative bacteria. Mol Microbiol 75:138–148
Swingle B, Markel E, Cartinhour S (2010) Oligonucleotide recombination: a hidden treasure. Bioeng Bugs 1:263–266. doi:10.4161/bbug.1.4.12098
Bryan A, Swanson MS (2011) Oligonucleotides stimulate genomic alterations of Legionella pneumophila. Mol Microbiol 80:231–247. doi:10.1111/j.1365-2958.2011.07573.x
Swingle B, Bao Z, Markel E, Chambers A, Cartinhour S (2010) Recombineering using RecTE from Pseudomonas syringae. Appl Environ Microbiol 76:4960–4968
Dutra BE, Sutera VA Jr, Lovett ST (2007) RecA-independent recombination is efficient but limited by exonucleases. Proc Natl Acad Sci U S A 104:216–221
Winans SC, Elledge SJ, Krueger JH, Walker GC (1985) Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol 161:1219–1221
Sawitzke JA, Costantino N, Li XT, Thomason LC, Bubunenko M, Court C, Court DL (2011) Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering. J Mol Biol 407:45–59. doi:S0022-2836(11)00059-3 [pii] 10.1016/j.jmb.2011.01.030
Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98:6742–6746
King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–307
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Choi KH, Kumar A, Schweizer HP (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397
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Swingle, B.M. (2013). Oligonucleotide Recombination Enabled Site-Specific Mutagenesis in Bacteria. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_9
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DOI: https://doi.org/10.1007/978-1-62703-293-3_9
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