A Broad Host Range Plasmid-Based Roadmap for ssDNA-Based Recombineering in Gram-Negative Bacteria

  • Tomás Aparicio
  • Víctor de LorenzoEmail author
  • Esteban Martínez-García
Part of the Methods in Molecular Biology book series (MIMB, volume 2075)


Recombineering is the use of phage recombination proteins to improve and facilitate bacterial genome engineering. Depending on the nature of the DNA template, double-stranded or single-stranded, the system needs three proteins (Gam, Exo, and Beta) or just one (Beta) to work properly. The use of this technique has been fundamental not only toward solving fundamental biological questions with reverse genetics but also for the generation of deep-engineered E. coli chassis strains. Unfortunately, the use of ssDNA recombineering is still limited to a narrow number of bacterial species. One of the reasons for that is the lack of proper recombinases to be efficiently used in different microorganisms and the lack of proper genetic tools to deliver and express this activity in a controlled way. Here, we describe a protocol to follow a simple workflow to identify, clone, and quantify the function of the selected recombinases in the organism of choice by cloning and expressing them in standardized broad host range plasmids. As an example of the method, we tested the use of the Ssr recombinase in P. putida EM42 by introducing a complete deletion of the target gene pyrF. The example shows how two parameters of the mutagenic oligo, i.e., length and phosphorothioate protection, affect the final outcome of the procedure.

Key words

Recombineering Recombinases ssDNA Conditional expression plasmids Genome editing Pseudomonas putida Synthetic biology 



This work was funded by the, HELIOS (BIO2015-66960-C3-2R) and SETH (RTI 2018-095584-B-C42) Projects of the Spanish Ministry of Science, the MADONNA (H2020-FET-OPEN-RIA-2017-1 (766975). BioRoboost (H2020-NMBP-BIO-CSA-2018-820699) and SYNBIO4FLAV (H2020-NMBP/0500-814650) Contracts of the European Union, and InGEMICS-CM (B2017/BMD-3691) contract of the Comunidad de Madrid (FSE, FECER). The authors declare that there is no conflict of interest. All the bacterial strains and plasmids described are available upon request.


  1. 1.
    Martínez-García E, de Lorenzo V (2011) Engineering multiple genomic deletions in gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 13(10):2702–2716CrossRefPubMedGoogle Scholar
  2. 2.
    Martínez-García E, de Lorenzo V (2012) Transposon-based and plasmid-based genetic tools for editing genomes of gram-negative bacteria. Methods Mol Biol 813:267–283CrossRefPubMedGoogle Scholar
  3. 3.
    Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180(8):2063–2071PubMedPubMedCentralGoogle Scholar
  4. 4.
    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):6640–6645CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ellis HM, Yu D, DiTizio T et al (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98(12):6742–6746CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wang HH, Isaacs FJ, Carr PA et al (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460(7257):894–898CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Gallagher RR, Li Z, Lewis AO et al (2014) Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA. Nat Protoc 9(10):2301–2316CrossRefPubMedGoogle Scholar
  8. 8.
    Binder S, Siedler S, Marienhagen J et al (2013) Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res 41(12):6360–6369CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    van Pijkeren JP, Britton RA (2012) High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res 40(10):e76CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat Methods 4(2):147–152CrossRefPubMedGoogle Scholar
  11. 11.
    Swingle B, Bao Z, Markel E et al (2010) Recombineering using RecTE from Pseudomonas syringae. Appl Environ Microbiol 76(15):4960–4968CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Isaacs FJ, Carr PA, Wang HH et al (2011) Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333(6040):348–353CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ricaurte DE, Martínez-García E, Nyerges A et al (2017) A standardized workflow for surveying recombinases expands bacterial genome editing capabilities. Microb Biotechnol 11(1):176–188. (In Press)CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Aparicio T, Jensen SI, Nielsen AT et al (2016) The Ssr protein (T1E_1405) from Pseudomonas putida DOT-T1E enables oligonucleotide-based recombineering in platform strain P. putida EM42. Biotechnol J 11(10):1309–1319CrossRefPubMedGoogle Scholar
  15. 15.
    Martínez-García E, Nikel PI, Aparicio T et al (2014) Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb Cell Factories 13:159CrossRefGoogle Scholar
  16. 16.
    Manoil C, Beckwith J (1985) TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A 82(23):8129–8133CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41(3):459–472CrossRefPubMedGoogle Scholar
  18. 18.
    Silva-Rocha R, Martínez-García E, Calles B et al (2013) The standard European vector architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res 41(Database issue):D666–D675CrossRefPubMedGoogle Scholar
  19. 19.
    Kessler B, de Lorenzo V, Timmis KN (1992) A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233(1–2):293–301CrossRefPubMedGoogle Scholar
  20. 20.
    Martínez-García E, Aparicio T, de Lorenzo V et al (2017) Engineering gram-negative microbial cell factories using transposon vectors. Methods Mol Biol 1498:273–293CrossRefPubMedGoogle Scholar
  21. 21.
    Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345CrossRefPubMedGoogle Scholar
  22. 22.
    Lopes A, Amarir-Bouhram J, Faure G et al (2010) Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Res 38:3952–3962CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410CrossRefPubMedGoogle Scholar
  24. 24.
    Martínez-García E, Aparicio T, Goñi-Moreno A et al (2015) SEVA 2.0: an update of the standard European vector architecture for de−/re-construction of bacterial functionalities. Nucleic Acids Res 43(Database issue):D1183–D1189CrossRefPubMedGoogle Scholar
  25. 25.
    Gawin A, Valla S, Brautaset T (2017) The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering. Microb Biotechnol 10(4):702–718CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Blatny JM, Brautaset T, Winther-Larsen HC et al (1997) Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon. Appl Environ Microbiol 63(2):370–379PubMedPubMedCentralGoogle Scholar
  27. 27.
    Mermod N, Ramos JL, Lehrbach PR et al (1986) Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria. J Bacteriol 167(2):447–454CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    O'Donovan GA, Neuhard J (1970) Pyrimidine metabolism in microorganisms. Bacteriol Rev 34(3):278–343PubMedPubMedCentralGoogle Scholar
  29. 29.
    Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197(2):345–346CrossRefPubMedGoogle Scholar
  30. 30.
    Galvao TC, de Lorenzo V (2005) Adaptation of the yeast URA3 selection system to gram-negative bacteria and generation of a ΔbetCDE Pseudomonas putida strain. Appl Environ Microbiol 71(2):883–892CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Timms AR, Steingrimsdottir H, Lehmann AR et al (1992) Mutant sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for spontaneous and ultraviolet light mutagenesis. Mol Gen Genet 232:89–96CrossRefPubMedGoogle Scholar
  32. 32.
    Yoshida H, Bogaki M, Nakamura M et al (1990) Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob Agents Chemother 34(6):1271–1272CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kureishi A, Diver JM, Beckthold B et al (1994) Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates. Antimicrob Agents Chemother 38(9):1944–1952CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Jatsenko T, Tover A, Tegova R et al (2010) Molecular characterization of Rif(r) mutations in Pseudomonas aeruginosa and Pseudomonas putida. Mutat Res 683(1–2):106–114CrossRefPubMedGoogle Scholar
  35. 35.
    Mythili E, Kumar KA, Muniyappa K (1996) Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking. Gene 182(1–2):81–87CrossRefPubMedGoogle Scholar
  36. 36.
    Babic I, Andrew SE, Jirik FR (1996) MutS interaction with mismatch and alkylated base containing DNA molecules detected by optical biosensor. Mutat Res 372(1):87–96CrossRefPubMedGoogle Scholar
  37. 37.
    Eckstein F (1985) Nucleoside phosphorothioates. Annu Rev Biochem 54:367–402CrossRefPubMedGoogle Scholar
  38. 38.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13):3406–3415CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Carr PA, Park JS, Lee YJ et al (2004) Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res 32(20):e162CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Tomás Aparicio
    • 1
  • Víctor de Lorenzo
    • 1
    Email author
  • Esteban Martínez-García
    • 1
  1. 1.Systems and Synthetic Biology ProgramCentro Nacional de Biotecnología (CNB-CSIC)MadridSpain

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