Recombineering Applications for the Mutational Analysis of Bacterial RNA-Binding Proteins and Their Sites of Action

  • Nara Figueroa-BossiEmail author
  • Lionello Bossi
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


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.

Key words

Recombineering Lambda red Site-directed mutagenesis Counterselection Markerless Scarless 



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.


  1. 1.
    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 97:6640–6645PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Murphy KC, Campellone KG, Poteete AR (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246:321–330PubMedCrossRefGoogle Scholar
  3. 3.
    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 97:5978–5983PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    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 421:171–199PubMedCrossRefGoogle Scholar
  5. 5.
    Thomason LC, Sawitzke JA, Li X, Costantino N, Court DL (2014) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 106:1.16.1–1.16.39CrossRefGoogle Scholar
  6. 6.
    Mosberg JA, Lajoie MJ, Church GM (2010) Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186:791–799PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    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 533:157–177PubMedCrossRefGoogle Scholar
  8. 8.
    Costantino N, Court DL (2003) Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc Natl Acad Sci U S A 100:15748–15753PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98:15264–15269PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    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 290:153–161PubMedCrossRefGoogle Scholar
  11. 11.
    Karlinsey JE (2007) λ-Red genetic engineering in Salmonella enterica serovar Typhimurium. Methods Enzymol 421:199–209PubMedCrossRefGoogle Scholar
  12. 12.
    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:e204PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Roberts MC (1996) Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol Rev 19:1–24PubMedCrossRefGoogle Scholar
  14. 14.
    Bochner BR, Huang HC, Schieven GL, Ames BN (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–933PubMedCentralPubMedGoogle Scholar
  15. 15.
    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 26:1864–1873PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    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 28:1239–1251PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1111PubMedCentralPubMedGoogle Scholar
  18. 18.
    Yang Q, Figueroa-Bossi N, Bossi L (2014) Translation enhancing ACA motifs and their silencing by a bacterial small regulatory RNA. PLoS Genet 10:e1004026PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Centre de Génétique MoléculaireCNRS UPR3404Gif-sur-YvetteFrance

Personalised recommendations