is a freelance medical writer in Mamaroneck, NY.
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It's one thing to conduct experiments in a controlled laboratory environment with well-characterized strains of microbes and other model organisms, and quite another to take one's work out into the real world where challenges include identifying unknown and uncharacterized organisms that may not grow under laboratory conditions, finding markers in nonmodel organisms with a genetic diversity that exceeds that of inbred strains, or having only rare or degraded specimens. To solve environmental problems through biotechnology may require the ability to handle large numbers of samples rapidly in real time, to develop new markers and detection technologies, and to conduct basic research under extreme conditions or at sites that can be accessed only with great difficulty.
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On the Surface
Alice C. Layton is a Research Associate Professor at The Center for Environmental Biotechnology (CEB), The University of Tennessee, Knoxville, TN. One area of Layton's research involves using bioluminescent reporter strains of Saccharomyces cerevisiae to detect low levels of estrogenic and androgenic compounds in environments, including waste stream treatment plants and farm animal manure. S. cerevisiae reporter strains have been engineered to express the lux operon of Photorhabdus luminescens and the flavin oxidoreductase from Vibrio harveyi. Layton and coworkers introduced estrogen and androgen response elements to generate reporters that respond to estrogenic and androgenic compounds.
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Another area of Layton's research applies real-time PCR technology to identify fecal sources in water. “The common theme of my research is to use available technology and apply it to environmental field situations,” Layton says. The advantage of using bioluminescent bioreporter strains is that more samples can be monitored more rapidly than with chemical analysis. The greater number of samples also provides a better profile of an area. One goal is to create an online monitoring system in situ, whereby waste effluents could be monitored continuously at the treatment plant. Others at CEB are adapting the bioluminescent reporter technology to microchips, so that rapid, high-throughput screening can be performed remotely in the field. The amount of bioluminescence from the yeast reporter strain is proportional to the amount of estrogenic or androgenic compound in the environment, and although the reaction does not precisely identify which specific steroid hormone is involved, follow-up chemical analysis can be performed. One of CEB's goals is to see this system become commercially available.
“It would nice,” she observes, “to have a company to either run the assays or to provide a kit that you could run yourself, containing preserved yeast to which you would just add the test sample.” She also envisions applying this technology to the detection of thyroid-reactive retinoids and other compounds in environmental samples. The reaction is based on the binding of an environmental contaminant to a cell-surface receptor or receptor-based nuclear response element, following which internalization of the signal turns on the luminescence reporter gene. Therefore, the system is applicable to any similarly acting receptor or nuclear response element-ligand reacting compound.
Digging DeeperSusan M. Pfiffner, Research Assistant Professor at the University of Tennessee and a colleague of Layton's at CEB, is interested in subterranean microbial communities. Mines in South Africa have provided access to environments as much as 3–4 km below the surface, allowing study of groundwater as well as organisms. Among other problems in working with deep surface organisms, isolation is difficult, the biomass is low, and the organisms may be barophilic. Phospholipid fatty acid analysis by chromatography works better for identification of the members of the microbial population than PCR, Pfiffner notes. Polar lipids present in viable populations are quickly cleaved by phospholipases into neutral lipids when organisms die, so relative amounts may be used to estimate current biomass and the past community. “We want to get an idea of who's there, how they get their energy, and how they use carbon,” she says. The composition of fatty acids may change when populations are stressed, exposed to a toxin, or have limited nutrient sources, reflecting cell membrane changes in response to the environment.
