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        Reporter Gene Expression for Monitoring Microorganisms in the Environment

        互联网

        665
        Reporter gene is a generic term that is used to describe genes whose expression is indicative of the presence of a particular cell, a specific event occurring within that cell, or a reflection of the expression of other, usually targeted genes of interest. For example, the presence of such a gene may make an organism standout to help in identification, as will be described in this chapter, or it could be that a gene is only expressed under certain conditions, e.g., during different developmental stages (1 ), environmental stresses (2 ,3 ) , or DNA repair as a result of induction of the SOS response to DNA damage (4 ). Naturally-occurring reporter genes can be used to avoid the need for genetic engineering by selecting a unique property of an organism and using it as the reporter. However, by far the commonest approach is to introduce a reporter gene into a cell to allow detection of a particular organism or to monitor its activity. The reporter gene can be located on a plasmid or to increase the stability of the gene within the cell it can be incorporated into the bacterial chromosome. There are a wide variety of reporter genes available, and all have particular properties that make them more or less useful depending on the application (Table 1 ). The choice of a particular reporter gene is made on the basis of the environment into which the organism is to be released, how the gene is to be detected, and what factors might interfere with the detection of the gene and/or its protein product.
        Table 1  Examples of Different Types of Reporter Genes

        Method of detection

        Source

        Reporter gene

        Gene product

        Mode of detection

        Reference

        Resistance

        Various plasmids

        Antibiotic resistance genes, e.g., catP amp

        For example, chloramphenicol acetyltransferase, β-lactamase

        Selective culture in presence of antibiotic, herbicide

        32

         

        Pseudomonas putida EEZ15

        Herbicide resistance

        Unknown

        or metal

        6

         

        Various plasmids

        Heavy metal resistance, e.g., merA

        For example, mercuric reductase

         

        5

        Fluorescence

        Fluorescent marine bacteria, e.g., Vibrio fischerii, V. harveyi, Photobacterium spp.

        lux

        Luciferase

        Scintillation counter, luminometry, CCD (chargecoupled device) camera,

        3,4,9,19–26

         

        Fire Fly

        luc

         

        X-ray film,

        33

         

        Click beetle

        rluc

         

        microscopy

        1

         

        Aequorea victoria (jellyfish) Renilla reniformis (sea pansy)

        gfP

        Green fluorescent protein (GFP)

        fluorescent microscopy, flow cytometry,

        15–18

         

        V. fischerii

        yfp

        Yellow fluorescent protein (YFP)

        confocal microscopy

        38

        Chromogenic

        E. coli, Bacillus stearothermophilus

        lacZY, BgaB

        β-Galactosidase

        Enzyme assay in presence of

        2,7,8,9 35

         

        E. coli

        gusA, uidA

        β-Glucuronidase

        appropriate substrate, e.g., X-Gal, catachol

        31

         

        TOL plasmid pWWO of Pseudomonas putida

        xylE

        Catechol 2,3-dioxygenase

        spectrophotometry, visually

        10–14

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