IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
artículos
Título:
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP
Autor/es:
SILVA JOAS; PIURI MARIANA; BROUSSARD GREGORY; MARINELLI LAURA; BASTOS GISELE; HIRATA ROSARIO; HATFULL GF; HIRATA MARIO
Revista:
FEMS MICROBIOLOGY LETTERS
Editorial:
WILEY-BLACKWELL PUBLISHING, INC
Referencias:
Lugar: Londres; Año: 2013 vol. 344 p. 166 - 172
ISSN:
0378-1097
Resumen:
Bacteriophage Recombineering of Electroporated DNA (BRED) has been mainly used for studying mycobacteriohage gene deletions and mutations. We aimed to insert the Hsp60-EGFP cassette (1143 bp) into D29 mycobacteriophage genome by BRED strategy and use the recombinant D29 for detection of mycobacterial cells. The cassette was successfully inserted and recombineered mycobacteriophages obtained. DNA sequencing of the cassette did not show any mutations even after several phage generations. M. smegmatis mc2 155 cells were infected with D29::Hsp60-EGFP (MOI10) and evaluated for a period of 6 h to detect EGFP under  fluorescence microscopy. After two-hours infection the majority of the fluorescent cells were lysed by the recombinant D29 phage. Further, the lysA gene was deleted to delay the lytic cycle of the recombinant phage. However, mycobacteriophage D29 ?lysA could not be recovered by either PCR or gene complementary assay, showing no viability. Using BRED technology we were able to insert a large segment encompassing the hsp60-EGPF into the genome of mycobacteriophage D29. This approach may be useful to construct novel reporter phages or to improve promising phage bacteriophages already developed.