Estamos trabajando para incorporar este artículo al repositorio
Consulte el artículo en la página del editor
Consulte la política de Acceso Abierto del editor


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


Documento: Artículo
Título:Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA
Autor:Marinelli, L.J.; Piuri, M.; Hatfull, G.F.
Filiación:Division of Dermatology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina
Laboratorio “Bacteriófagos y Aplicaciones Biotecnológicas”, Departamento de Química Biológica, FCEyN, UBA, Ciudad Universitaria, Ciudad Autónoma de Buenos Aires, Argentina
Department of Biological Sciences and Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA, United States
Palabras clave:BRED; Electroporation; Mycobacteria; Mycobacteriophage; Recombineering; bacteriophage DNA; genomic DNA; oligonucleotide; Rac protein; recombinant protein; RecT protein; bacteriophage recombineering of electroporated DNA; bacterium culture; electroporation; genetic engineering; genetic manipulation; genetic recombination; methodology; mutant; mycobacteriophage; Mycobacterium smegmatis; natural science; nonhuman; polymerase chain reaction; prophage; recombineering
Página de inicio:69
Página de fin:80
Título revista:Methods in Molecular Biology
Título revista abreviado:Methods Mol. Biol.


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


---------- APA ----------
Marinelli, L.J., Piuri, M. & Hatfull, G.F. (2019) . Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA. Methods in Molecular Biology, 1898, 69-80.
---------- CHICAGO ----------
Marinelli, L.J., Piuri, M., Hatfull, G.F. "Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA" . Methods in Molecular Biology 1898 (2019) : 69-80.
---------- MLA ----------
Marinelli, L.J., Piuri, M., Hatfull, G.F. "Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA" . Methods in Molecular Biology, vol. 1898, 2019, pp. 69-80.
---------- VANCOUVER ----------
Marinelli, L.J., Piuri, M., Hatfull, G.F. Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA. Methods Mol. Biol. 2019;1898:69-80.