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

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

Abstract

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 

Notes

Acknowledgments

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.

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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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