title: Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.
abstract: 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 mc(2) 155 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.
authors: Laboratory of Applied Molecular Biology and Pharmacogenomics, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil. joaslucas@usp.br
authors: Joas L
authors: JL
authors: da Silva
authors: Mariana
authors: M
authors: Piuri
authors: Gregory
authors: G
authors: Broussard
authors: Laura J
authors: LJ
authors: Marinelli
authors: Gisele M
authors: GM
authors: Bastos
authors: Rosario D C
authors: RD
authors: Hirata
authors: Graham F
authors: GF
authors: Hatfull
authors: Mario H
authors: MH
authors: Hirata
mesh_terms: D018274:Electroporation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008242:Lysogeny; D009160:Mycobacteriophages / Q000235:genetics* / Q000502:physiology; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism / Q000821:virology*; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins