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High-Efficiency Multi-site Genomic Editing (HEMSE) Made Easy

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Recombineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2479))

Abstract

The ability to engineer bacterial genomes in an efficient way is crucial for many bio-related technologies. Single-stranded (ss) DNA recombineering technology allows to introduce mutations within bacterial genomes in a very simple and straightforward way. This technology was initially developed for E. coli but was later extended to other organisms of interest, including the environmentally and metabolically versatile Pseudomonas putida. The technology is based on three pillars: (1) adoption of a phage recombinase that works effectively in the target strain, (2) ease of introduction of short ssDNA oligonucleotide that carries the mutation into the bacterial cells at stake and (3) momentary suppression of the endogenous mismatch repair (MMR) through transient expression of a dominant negative mutL allele. In this way, the recombinase protects the ssDNA and stimulates recombination, while MutLE36KPP temporarily inhibits the endogenous MMR system, thereby allowing the introduction of virtually any possible type of genomic edits. In this chapter, a protocol is detailed for easily performing recombineering experiments aimed at entering single and multiple changes in the chromosome of P. putida. This was made by implementing the workflow named High-Efficiency Multi-site genomic Editing (HEMSE), which delivers simultaneous mutations with a simple and effective protocol.

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Acknowledgments

This work was funded by the SETH (RTI2018-095584-B-C42) (MINECO/FEDER), SyCoLiM (ERA-COBIOTECH 2018—PCI2019-111859-2) Projects of the Spanish Ministry of Science and Innovation, The MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), SynBio4Flav (H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650) and MIX-UP (MIX-UP H2020-BIO-CN-2019-870294) Contracts of the European Union and the InGEMICS-CM (S2017/BMD-3691) and BIOSINT-CM (Y2020/TCS-6555) Projects of the Comunidad de Madrid—European Structural and Investment Funds—(FSE, FECER). The authors declare that there is no conflict of interest. The bacterial strains and plasmids described are available upon request.

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Authors and Affiliations

  1. Systems Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain

    Tomás Aparicio, Víctor de Lorenzo & Esteban Martínez-García

Authors
  1. Tomás Aparicio
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  2. Víctor de Lorenzo
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  3. Esteban Martínez-García
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Corresponding author

Correspondence to Víctor de Lorenzo .

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Editors and Affiliations

  1. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA

    Christopher R. Reisch

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Aparicio, T., de Lorenzo, V., Martínez-García, E. (2022). High-Efficiency Multi-site Genomic Editing (HEMSE) Made Easy. In: Reisch, C.R. (eds) Recombineering. Methods in Molecular Biology, vol 2479. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2233-9_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2233-9_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2232-2

  • Online ISBN: 978-1-0716-2233-9

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