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.

CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages

“…The Bacteriophage Recombineering of Electroporated DNA (BRED) technique was recently developed to generate specific mutations, insertions, deletions and gene replacement in lytic mycobacteriophage (4,6). BRED exploits a phage recombinase to induce homologous recombination (5,7–10) between a phage genome and a template, which must be electroporated into the cell (5).…”

Molecular Genetics of Mycobacteriophages

“…First, a derivative of phage L5 that carries the firefly luciferase gene was constructed by crossing over from a plasmid and identifying the recombinants by hybridization (165); these infect both fast- and slow-growing mycobacteria (195). Second, a reporter phage derivative of D29 has been constructed carrying green fluorescent protein (GFP) using recombineering (196). These approaches are more directed than using shuttle phasmids but contribute toward a suite of strategies for using phages to deliver DNA efficiently to mycobacterial hosts.…”

“…Reporter phages have been built using a variety of phage genomes including TM4, D29, and L5, all of which can infect both M. smegmatis and M. tuberculosis (165, 188, 196, 226); the TM4 platform may offer some advantages because of its ability to infect stationary-phase cells, a phenomenon that is dependent on the peptidoglycan hydrolytic motif embedded within the phage Tapemeasure protein (106). The main difference in the choice of the reporter is the detection method used.…”

Phage-based detection of bacterial pathogens

“…81 The hsp60 promoter and EGFP cassette also showed detectable uorescence levels when transferred to mycobacteriophage D29 and used to infect M. smegmatis. 82 Another study showed that addition of a Strep-tag II fusion to the phAE87:hsp60-EGFP gp9 C-terminus enabled affinity purication of host bacterial complexes. 83 One study involved the development of a uorescent mycobacteriophage assay with a 100 fold increase in uorescence per cell over phAE87:hsp60-EGFP.…”