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Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

John Sanseverino,^1,2 Rakesh K. Gupta,^1, Alice C. Layton,^1,2 Stacey S. Patterson,^1, Steven A. Ripp,^1,2 Leslie Saidak,¹ Michael L. Simpson,^1,3 T. Wayne Schultz,^1,4 and Gary S. Sayler^1,2*

The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, Tennessee 37996,¹ The Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996,² Molecular Scale Engineering and Nanoscale Technologies Research Group, Oak Ridge National Laboratory, P.O. Box 2008, MS 6006, Oak Ridge, Tennessee 37831,³ Department of Comparative Medicine, The University of Tennessee, Knoxville, Tennessee 37996⁴

Received 9 November 2004/ Accepted 11 March 2005

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17ß-estradiol over the concentration range of 1.2 x 10^–8 through 5.6 x 10^–12 M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) x 10^–10 and (2.4 ± 1.0) x 10^–10 M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10^–11 to 2.8 x 10^–9 M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.

Present address: Department of Microbiology, RLA College, University of Delhi, New Delhi 110021, India.

Present address: College of Marine Science, University of South Florida, St. Petersburg, FL 33701.

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