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

Authors

  • Joas L. da Silva,

    Corresponding author
    • Laboratory of Applied Molecular Biology and Pharmacogenomics, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil
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  • Mariana Piuri,

    1. Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Buenos Aires, Argentina
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  • Gregory Broussard,

    1. Department of Biological Sciences, Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA, USA
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  • Laura J. Marinelli,

    1. Department of Biological Sciences, Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA, USA
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  • Gisele M. Bastos,

    1. Laboratory of Applied Molecular Biology and Pharmacogenomics, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil
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  • Rosario D.C. Hirata,

    1. Laboratory of Applied Molecular Biology and Pharmacogenomics, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil
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  • Graham F. Hatfull,

    1. Department of Biological Sciences, Pittsburgh Bacteriophage Institute, University of Pittsburgh, Pittsburgh, PA, USA
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  • Mario H. Hirata

    1. Laboratory of Applied Molecular Biology and Pharmacogenomics, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil
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Correspondence: Joas L. da Silva, Laboratory of Applied Molecular Biology and Pharmacogenomics, School of Pharmaceutical Sciences, University of Sao Paulo, Av. Lineu Prestes 580, B-17, 05508-900, São Paulo, SP, Brazil. Tel./fax: +55 11 3091 3660; e-mail: joaslucas@usp.br

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 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.

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