Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP

• Autores: da Silva, J.L.; Piuri, M.; Broussard, G.; J. Marinelli, L.; Bastos, G.M.; Hirata, R.D.C.; Hatfull, G.F.; Hirata, M.H.
• Año de publicación: 2013
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: FEMS Microbiol. Lett. 2013;344(2):166-172
• Materia: Bacteriophage; Green fluorescent protein; Mycobacterium; Recombineering; DNA; enhanced green fluorescent protein; genomic DNA; analytic method; article; bacteriophage; Bacteriophage Recombineering of Electroporated DNA technology; cytolysis; DNA sequence; gene cassette; gene deletion; gene mutation; nonhuman; point mutation; priority journal; protein expression; Electroporation; Genes, Reporter; Green Fluorescent Proteins; Lysogeny; Mycobacteriophages; Mycobacterium smegmatis; Promoter Regions, Genetic; Sequence Deletion; Mycobacterium; Mycobacterium smegmatis
• Institución: Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales

Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd.
Fil:Piuri, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.