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Title: TRANSFORMATION OF ESCHERICHIA COLI K-12 WITH A HIGH COPY PLASMID ENCODING THE GREEN FLUORESCENT PROTEIN OF AEQUOREA VICTORIA RESULTS IN REDUCED FITNESS IN THE FORM OF SLOWER GROWTH

Author
item Oscar, Thomas
item DULAL, KALPANA - UMES
item BOUCAUD, DWAYNE - UMES

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2005
Publication Date: 2/1/2006
Citation: Oscar, T.P., Dulal, K., Boucaud, D. 2006. Transformation of escherichia coli k-12 with a high copy plasmid encoding the green fluorescent protein of aequorea victoria results in reduced fitness in the form of slower growth. Applied and Environmental Microbiology. Vol. 69. No.2. pages 276-281.

Interpretive Summary: A data gap identified in recent risk assessments for food pathogens is models that predict the behavior of pathogens in naturally contaminated food. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a widely used biomarker for following growth, death and dissemination of a single strain of bacteria in water, food and environmental systems. However, before a GFP pathogen can be validly used to develop a model in food with competitive microflora, it must be demonstrated that behavior of the GFP pathogen is the same as the strain from which it was made. Many reports indicate GFP does not alter behavior of bacteria but a few recent reports indicate GFP can induce fitness problems, such as slower growth. In the current study, E. coli bacteria that were genetically engineered to produce GFP grew slower than the strain from which they were made. However, the fitness problem resulted from the method of DNA introduction rather than production of GFP. Alternative methods of introducing GFP into E. coli may lead to valid strains for modeling behavior in naturally contaminated food.

Technical Abstract: The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker in eucaryotic and prokaryotic cells and has potential for developing predictive models for behavior of single strains of bacteria in naturally contaminated food and environmental samples. However, constitutive production of GFP in bacteria can result in reduced fitness in the form of slower growth. Consequently, a high copy plasmid with gfp under the control of a tetracycline inducible promoter was introduced into Escherichia coli K-12. To validate the GFP strain of E. coli K-12 for predictive modeling studies, growth kinetics of the parent and GFP strain were compared at 10, 25 and 40 C on sterilized cooked chicken breast meat (sBM). Although gfp expression was not induced during growth on sBM, maximum specific growth rate of the GFP strain was reduced (P < 0.05), regardless of incubation temperature. When growth kinetics were compared in BHI broth at 40 C, maximum specific growth rate and maximum population density were reduced (P < 0.05) to the same extent in the absence and presence of tetracycline, which induced the expression of gfp. These results indicated that the presence of the high copy plasmid introduced a fitness problem in E. coli K-12 in the form of slower growth and reduced cell yield that was independent of GFP production. Thus, use of a low copy plasmid or insertion of a single copy of gfp into the chromosome may be required to avoid fitness problems in GFP bacteria.