Development of a bioluminescence-based detection system for a genetically modified microorganism
A molecular-based monitoring system was developed for the detection and enumeration of a genetically modified microorganism, based on bacterial bioluminescence. Plasmids containing various cassettes of the genes encoding bacterial bioluminescence were introduced into the model organism, Escherichia coli. Bioluminescence was monitored during growth using luminometry. Light output varied with host strain and plasmid construct. Those strains containing the entire lux operon exhibited similar bioluminescence profiles to Vibrio fischeri, with induction of light production being dependent on cell density. Escherichia coli strains containing a truncated lux operon showed partial induction of luminescence with growth. The most appropriate constructs for environmental detection were those only containing the genes coding for the functional subunits of luciferase (luxAB) under the control of a constitutive promoter. Light output from these strains was proportional to biomass concentration during growth. Light output in all strains tested decreased during stationary phase, due to a decrease in metabolic activity. Light output from luminescent strains was investigated after inoculation into soil. For a particular cell density, there was a 10-fold decrease in luminescence when the cells were suspended in a soil slurry compared to a liquid culture. Detection of lux-modified E. coli ranged from 102-103 cells (g soil)-1, depending on the host strain and plasmid used. Luminometry was compared to traditional methods for the estimation of microbial activity, using both L-broth and sterile soil, and was found to measure the actual activity of lux-modified cells in systems. Survival and activity was greatly decreased by competition, due to the presence of the indigenous soil population. The addition of substrate enhanced the survival and activity of lux-modified E. coli in non-sterile soil. Increasing matric stress had a deleterious effect on the survival and luminescence of E. coli in sterile soil. Although both respiratory and luminescence activity were enhanced due to the addition of glucose to the soil, both parameters, nonetheless decreased with increasing matric stress. The differences in response to varying matric stress were highlighted through differences in the actual and potential activities demonstrated by E. coli. It is proposed that the luminescence-based techniques developed in this thesis will contribute to a suite of detection methodologies for the assessment of risk of environmental introduction of genetically modified microorganisms.