Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria

Authors

  • Riccardo Tombolini,

    1. Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden
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  • Annika Unge,

    1. Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden
    2. NSF Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, USA
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  • Mary Ellen Davey,

    1. NSF Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, USA
    2. Department of Microbiology, Michigan State University, East Lansing, MI 48824, USA
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  • Frans J de Bruijn,

    1. NSF Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, USA
    2. Department of Microbiology, Michigan State University, East Lansing, MI 48824, USA
    3. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
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  • Janet K Jansson

    Corresponding author
    1. Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden
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Corresponding author. Tel.: +46 (8) 16 2469; Fax: +46 (8) 15 3679; E-mail: janet@biokemi.su.se

Abstract

The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria was assembled into an expression cassette for bacteria by fusing it to the T7 gene10 ribosome binding site and the strong, constitutive promoter PpsbA from Amaranthus hybridus. By using Tn5-based transposon delivery systems, Pseudomonas fluorescens bacteria were chromosomally tagged with gfp. We demonstrate that expression of a single copy gfp gene is sufficient to permit the visualization of bacteria by epifluorescence and laser confocal microscopy and detection by flow cytometry. The green fluorescent phenotype was detectable in all growth phases even under nutrient-limited conditions.

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