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Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA

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Bacteriophages

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1898))

Abstract

We describe a recombineering-based method for the genetic manipulation of lytically replicating bacteriophages, focusing on mycobacteriophages. The approach utilizes recombineering-proficient strains of Mycobacterium smegmatis and employs a cotransformation strategy with purified phage genomic DNA and a mutagenic substrate, which selects for only those cells that are competent to take up DNA. The cotransformation method, combined with the high rates of recombination obtained in M. smegmatis recombineering strains, allows for the efficient and rapid generation of bacteriophage mutants.

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Acknowledgments

The authors wish to thank Dr. Rebekah Dedrick for generously providing a critical reading of the protocol and for helpful comments and discussions.

Author information

Authors and Affiliations

  1. Division of Dermatology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Laura J. Marinelli

  2. Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina

    Mariana Piuri

  3. Laboratorio “Bacteriófagos y Aplicaciones Biotecnológicas”, Departamento de Química Biológica, FCEyN, UBA, Ciudad Universitaria, Ciudad Autónoma de Buenos Aires, Argentina

    Mariana Piuri

  4. Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA, USA

    Graham F. Hatfull

Authors
  1. Laura J. Marinelli
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  2. Mariana Piuri
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  3. Graham F. Hatfull
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Editor information

Editors and Affiliations

  1. Department of Genetics and Genome Biology, University of Leicester, Leicester, UK

    Martha R. J. Clokie

  2. Departments of Food Science, and Pathobiology, University of Guelph, Guelph, ON, Canada

    Andrew Kropinski

  3. Laboratory of Gene Technology, KU Leuven, Leuven, Belgium

    Rob Lavigne

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Marinelli, L.J., Piuri, M., Hatfull, G.F. (2019). Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA. In: Clokie, M., Kropinski, A., Lavigne, R. (eds) Bacteriophages. Methods in Molecular Biology, vol 1898. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8940-9_6

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  • DOI: https://doi.org/10.1007/978-1-4939-8940-9_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8939-3

  • Online ISBN: 978-1-4939-8940-9

  • eBook Packages: Springer Protocols

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