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

  • Laura J. Marinelli
  • Mariana Piuri
  • Graham F. Hatfull
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

BRED Recombineering Electroporation Mycobacteria Mycobacteriophage 

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Laura J. Marinelli
    • 1
  • Mariana Piuri
    • 2
    • 3
  • Graham F. Hatfull
    • 4
  1. 1.Division of Dermatology, Department of Medicine, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUSA
  2. 2.Departamento de Química Biológica, Facultad de Ciencias Exactas y NaturalesUniversidad de Buenos Aires, IQUIBICEN-CONICETBuenos AiresArgentina
  3. 3.Laboratorio “Bacteriófagos y Aplicaciones Biotecnológicas”, Departamento de Química Biológica, FCEyN, UBACiudad UniversitariaCiudad Autónoma de Buenos AiresArgentina
  4. 4.Department of Biological Sciences and Pittsburgh Bacteriophage InstituteUniversity of PittsburghPittsburghUSA

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