News
Living sensors for saccharides Call them bacterial beacons. The E. colt genetically engineered by Sylvia Daunert and colleagues at the University of KentuckyLexington detect the monosaccharide L-arabinose in their surroundings and fluoresce in response (Anal. Chetn. 1999, 71, 763-68). "We wanted to develop bacteria-based sensing systems to take advantage of the tremendous selectivity and sensitivity that these natural systems have to offer," Daunert says. "It is very difficult to discriminate between sugars and to have selectivity for stereoisomers, but these bacterial systems provide a very nice way to do it." Because monosaccharides and their stereoisomers are very similar, most detection systems need a separation step—chromatographic separation or capillary zone electrophoresis—to achieve selectivity. But Daunert's bacterial sensors distinguish L-arabinose from sugars such as fructose, glucose, and D-arabinose without such a step. Instead, the sensors' specificity is conferred by the protein AraC, which binds to L-arabinose and controls a naturally occurring cluster of genes called the araBAD operon. When L-arabinose is present AraC is the operon's activator enabling protein production But when L-arabinose is absent AraC is a repressor preventing production AraC achieves this control by binding to various regions of the operon's promoter. The promoter is the DNA sequence where RNA polymerase binds, initiating transcription of messenger RNA (mRNA), which gets translated into protein. When there is no L-arabinose, AraC binds to the promot-
er's initiator region at the same time it binds to the operator region a short distance away. This creates a loop in the DNA, occluding the binding site for RNA polymerase and preventing protein production. But when there is L-arabinose, it binds to AraC and changes the protein's conformation. AraC detaches from the operator region, eliminating the loop and allowing transcription of mRNA. In Daunert's L-arabinose sensors, the operon's usual cluster of genes—araB, araA, and araD—are replaced by the green fluorescent protein (GFP) from jellyfish. The researchers chose GFP because it is not toxic at high levels and, unlike some reporter genes, does not require addition of a substrate. "You just shine light, and you see emission," Daunert says. "It is very easy to use." Another advantage of the system, she says, is that it provides natural amplification of the signal—that is, the binding of AraC to L-arabinose. One stage of amplification occurs during transcription, when multiple copies of the message are made. A second stage occurs during translation, when multiple copies of the GFP protein are made. Nonetheless, the system is quantitative, Daunert says. She and her colleagues have shown that the amount of fluorescence is related to the concentration of L-arabinose in the bacteria's surroundings and to the induction time—the amount of time that elapses before fluorescence measurements are taken. With 5-min induction times, L-arabinose levels as low as 1 x 10~5 M have been detected. With 60-min induction times, the levels have been as low as 5 x 10~7 M.
BUSINESS
Perkin-Elmer to sell Analytical Instruments Division In a move that comes as no surprise, Perkin-Elmer announced in January 1999 its intention to find a buyer for its Analytical Instruments Division. This action is a result of a strategic review of the division that was begun in September 1998. PerkinElmer will report the financial results for the division as a discontinued operation for the quarter ending December 31,1998. According to a statement by Tony L. White, chairman, president, and chief executive officer of Perkin-Elmer, the sale will help meet two "strategic goals" of strengthening the company's position as a "preeminent supplier" to the life sciences marketplace and of "determining viable and sustainable growth opportunities for Analytical Instruments".
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Analytical Chemistry News & Features, March 1, 1999
Expression of green fluorescent protein (GFP), encoded by the gene gtpuv, is regulated by the protein AraC. When L-arabinose is absent (top), AraC binds to the initiator region (aral,) and the operator region (ara02), creating a DNA loop that prevents RNA polymerase from binding. When L-arabinose is present (bottom), it binds to AraC, changing the protein's conformation. AraC detaches from the operator region, eliminating the loop and allowing transcription and translation.
The signals can be measured by loading samples into a standard fluorescence cuvette or by using an in situ fiber-optic probe, Daunert explains. Designed for continuous monitoring, the probe can track analyte levels in effluent streams, bioreactors, or odier environments by trapping 100-uL samples behind a dialysis membrane at the tip of the fiber. "We chose L-arabinose as a model to show that we can detect sugars—as well as metals, which we detected in the past—and to show that we can use natural systems to achieve tremendous selectivity," Daunert says. The next step will be to multiplex such a system and detect several analytes at once, she says. "We believe these bacteriabased systems have applicability to a wide variety of analytes," says Daunert. "The goal is to have various GFP mutants or reporter genes emit at different wavelengths and sense different analytes. This would give you array detection, which would [really] let you see how a system is doing." Elizabeth Zubritsky
