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

  • Laura J. Marinelli
  • Mariana Piuri
  • Graham F. Hatfull
Part of the Methods in Molecular Biology book series (MIMB, volume 1898)


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 



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


  1. 1.
    Hatfull GF, Hendrix RW (2011) Bacteriophages and their genomes. Curr Opin Virol 1:298–303CrossRefGoogle Scholar
  2. 2.
    Katsura I (1976) Isolation of lambda prophage mutants defective in structural genes: their use for the study of bacteriophage morphogenesis. Mol Gen Genet MGG 148:31CrossRefGoogle Scholar
  3. 3.
    Katsura I, Hendrix RW (1984) Length determination in bacteriophage lambda tails. Cell 39:691CrossRefGoogle Scholar
  4. 4.
    Selick HE, Kreuzer KN, Alberts BM (1988) The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome. J Biol Chem 263:11336PubMedGoogle Scholar
  5. 5.
    Struthers-Schlinke JS, Robins WP, Kemp P, Molineux IJ (2000) The internal head protein Gp16 controls DNA ejection from the bacteriophage T7 virion. J Mol Biol 301:35CrossRefGoogle Scholar
  6. 6.
    Moak M, Molineux IJ (2000) Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection. Mol Microbiol 37:345CrossRefGoogle Scholar
  7. 7.
    Oppenheim AB, Rattray AJ, Bubunenko M, Thomason LC, Court DL (2004) In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides. Virology 319:185CrossRefGoogle Scholar
  8. 8.
    Murray NE (2006) The impact of phage lambda: from restriction to recombineering. Biochem Soc Trans 34:203CrossRefGoogle Scholar
  9. 9.
    Piuri M, Hatfull GF (2006) A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells. Mol Microbiol 62:1569CrossRefGoogle Scholar
  10. 10.
    Martel B, Moineau S (2014) CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res 42:9504CrossRefGoogle Scholar
  11. 11.
    van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat Methods 4:147CrossRefGoogle Scholar
  12. 12.
    van Kessel JC, Hatfull GF (2008) Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets. Mol Microbiol 67:1094CrossRefGoogle Scholar
  13. 13.
    van Kessel JC, Hatfull GF (2008) Mycobacterial recombineering. Methods Mol Biol 435:203CrossRefGoogle Scholar
  14. 14.
    van Kessel JC, Marinelli LJ, Hatfull GF (2008) Recombineering mycobacteria and their phages. Nat Rev Microbiol 6:851CrossRefGoogle Scholar
  15. 15.
    Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361CrossRefGoogle Scholar
  16. 16.
    Little JW (1967) An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction. J Biol Chem 242:679PubMedGoogle Scholar
  17. 17.
    Joseph JW, Kolodner R (1983) Exonuclease VIII of Escherichia coli. II. Mechanism of action. J Biol Chem 258:10418PubMedGoogle Scholar
  18. 18.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640CrossRefGoogle Scholar
  19. 19.
    Yu D et al (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97:5978CrossRefGoogle Scholar
  20. 20.
    Hall SD, Kolodner RD (1994) Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein. Proc Natl Acad Sci U S A 91:3205CrossRefGoogle Scholar
  21. 21.
    Kolodner R, Hall SD, Luisi-DeLuca C (1994) Homologous pairing proteins encoded by the Escherichia coli recE and recT genes. Mol Microbiol 11:23CrossRefGoogle Scholar
  22. 22.
    Noirot P, Kolodner RD (1998) DNA strand invasion promoted by Escherichia coli RecT protein. J Biol Chem 273:12274CrossRefGoogle Scholar
  23. 23.
    Li Z, Karakousis G, Chiu SK, Reddy G, Radding CM (1998) The beta protein of phage lambda promotes strand exchange. J Mol Biol 276:733CrossRefGoogle Scholar
  24. 24.
    Rybalchenko N, Golub EI, Bi B, Radding CM (2004) Strand invasion promoted by recombination protein beta of coliphage lambda. Proc Natl Acad Sci U S A 101:17056CrossRefGoogle Scholar
  25. 25.
    Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063PubMedPubMedCentralGoogle Scholar
  26. 26.
    Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123CrossRefGoogle Scholar
  27. 27.
    Muyrers JP, Zhang Y, Testa G, Stewart AF (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555CrossRefGoogle Scholar
  28. 28.
    Murphy KC, Campellone KG, Poteete AR (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246:321CrossRefGoogle Scholar
  29. 29.
    Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98:6742CrossRefGoogle Scholar
  30. 30.
    Lee EC et al (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56CrossRefGoogle Scholar
  31. 31.
    Muyrers JP, Zhang Y, Stewart AF (2001) Techniques: recombinogenic engineering—new options for cloning and manipulating DNA. Trends Biochem Sci 26:325CrossRefGoogle Scholar
  32. 32.
    Marinelli LJ et al (2008) BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One 3:e3957CrossRefGoogle Scholar
  33. 33.
    Marinelli LJ, Hatfull GF, Piuri M (2012) Recombineering: a powerful tool for modification of bacteriophage genomes. Bacteriophage 2:5CrossRefGoogle Scholar
  34. 34.
    Payne K, Sun Q, Sacchettini J, Hatfull GF (2009) Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase. Mol Microbiol 73:367CrossRefGoogle Scholar
  35. 35.
    Catalao MJ, Gil F, Moniz-Pereira J, Pimentel M (2010) The mycobacteriophage Ms6 encodes a chaperone-like protein involved in the endolysin delivery to the peptidoglycan. Mol Microbiol 77:672CrossRefGoogle Scholar
  36. 36.
    Catalao MJ, Milho C, Gil F, Moniz-Pereira J, Pimentel M (2011) A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6. PLoS One 6:e20515CrossRefGoogle Scholar
  37. 37.
    Catalao MJ, Gil F, Moniz-Pereira J, Pimentel M (2011) Functional analysis of the holin-like proteins of mycobacteriophage Ms6. J Bacteriol 193:2793CrossRefGoogle Scholar
  38. 38.
    Savinov A, Pan J, Ghosh P, Hatfull GF (2012) The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication. Gene 495:42CrossRefGoogle Scholar
  39. 39.
    Jacobs-Sera D et al (2012) On the nature of mycobacteriophage diversity and host preference. Virology 434:187CrossRefGoogle Scholar
  40. 40.
    Dedrick RM et al (2013) Functional requirements for bacteriophage growth: gene essentiality and expression in mycobacteriophage Giles. Mol Microbiol 88:577CrossRefGoogle Scholar
  41. 41.
    da Silva JL et al (2013) Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP. FEMS Microbiol Lett 344:166CrossRefGoogle Scholar
  42. 42.
    Piuri M, Rondon L, Urdaniz E, Hatfull GF (2013) Generation of affinity-tagged fluoromycobacteriophages by mixed assembly of phage capsids. Appl Environ Microbiol 79:5608CrossRefGoogle Scholar
  43. 43.
    Feher T, Karcagi I, Blattner FR, Posfai G (2012) Bacteriophage recombineering in the lytic state using the lambda red recombinases. Microb Biotechnol 5:466CrossRefGoogle Scholar
  44. 44.
    Shin H, Lee JH, Yoon H, Kang DH, Ryu S (2014) Genomic investigation of lysogen formation and host lysis systems of the Salmonella temperate bacteriophage SPN9CC. Appl Environ Microbiol 80:374CrossRefGoogle Scholar
  45. 45.
    Swaminathan S et al (2001) Rapid engineering of bacterial artificial chromosomes using oligonucleotides. Genesis 29:14CrossRefGoogle Scholar

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

Personalised recommendations