Abstract
Genetics remains a powerful tool to study structure–function relationships in proteins and RNA. Structural elements important for the biological activity of these molecules can be dissected through the isolation of mutations and analysis of their effects on the mechanism under study. In suitable model organisms, this approach can greatly benefit from the ability to introduce mutations directly in the chromosomal context in ways that do not perturb neighboring sequences. Methods for performing such “markerless” site-directed chromosomal mutagenesis in bacteria have been developed in recent years. One such technique, used routinely in our laboratory, is described here.
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References
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 97:6640–6645
Murphy KC, Campellone KG, Poteete AR (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246:321–330
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
Sawitzke JA, Thomason LC, Costantino N, Bubunenko M, Datta S, Court DL (2007) Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol 421:171–199
Thomason LC, Sawitzke JA, Li X, Costantino N, Court DL (2014) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 106:1.16.1–1.16.39
Mosberg JA, Lajoie MJ, Church GM (2010) Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186:791–799
Sawitzke JA, Thomason LC, Bubunenko M, Li X, Costantino N, Court DL (2013) Recombineering: highly efficient in vivo genetic engineering using single-strand oligos. Methods Enzymol 533:157–177
Costantino N, Court DL (2003) Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc Natl Acad Sci U S A 100:15748–15753
Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98:15264–15269
Ellermeier CD, Janakiraman A, Slauch JM (2002) Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria. Gene 290:153–161
Karlinsey JE (2007) λ-Red genetic engineering in Salmonella enterica serovar Typhimurium. Methods Enzymol 421:199–209
Li XT, Thomason LC, Sawitzke JA, Costantino N, Court DL (2013) Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res 41:e204
Roberts MC (1996) Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol Rev 19:1–24
Bochner BR, Huang HC, Schieven GL, Ames BN (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–933
Bossi L, Schwartz A, Guillemardet B, Boudvillain M, Figueroa-Bossi N (2012) A role for Rho-dependent polarity in gene regulation by a noncoding small RNA. Genes Dev 26:1864–1873
Figueroa-Bossi N, Schwartz A, Guillemardet B, D’Heygere F, Bossi L, Boudvillain M (2014) RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination. Genes Dev 28:1239–1251
Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1111
Yang Q, Figueroa-Bossi N, Bossi L (2014) Translation enhancing ACA motifs and their silencing by a bacterial small regulatory RNA. PLoS Genet 10:e1004026
Acknowledgments
We would like to thank Kelly Hughes and Fabienne Chevance for initially encouraging us to develop the protocol described here. This work was supported by French National Research Agency (ANR) ANR-13-BSV3-0005-01.
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Figueroa-Bossi, N., Bossi, L. (2015). Recombineering Applications for the Mutational Analysis of Bacterial RNA-Binding Proteins and Their Sites of Action. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 1259. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2214-7_7
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DOI: https://doi.org/10.1007/978-1-4939-2214-7_7
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