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.
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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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