1. 10 5 10 4 10 3 10 2 10 1 0 E. coli cfu/mL Genetic engineering of a yeast-based bioluminescent bioreporter to detect dioxins. Alexandra Lynn Department of Microbiology, Center for Environmental Biotechnology, University of Tennessee, Knoxville Abstract Conclusions The dioxin-responsive strain: Bioluminescent Yeast Aryl Hydrocarbon Screen Light Dioxin Due to increasing concerns over exposure of humans and wildlife to harmful chemicals in the environment, many assays have been developed for their rapid detection. One set of chemicals that is beginning to receive more attention are those that interact with the human aryl hydrocarbon receptor system. Chemicals like dioxins and polycyclic aromatic hydrocarbons (PAHs) are known to be extremely toxic to humans, ultimately as mutagens and carcinogens, so determining if they are present in the environment is very important. A bioluminescent yeast bioreporter was created for the detection of chemicals that interact with the human aryl hydrocarbon receptor response system. A yeast strain (from the American Type Culture Collection, ATCC) that contains the human aryl hydrocarbon receptor protein on its genome (Saccharomyces cerevisiae YCM3) was genetically engineered to produce bioluminescence in the presence of dioxins and any other aryl hydrocarbon mimics. Using PCR amplification and ligation of the aryl hydrocarbon response elements (XREs) from humans in between two promoters (GPD and ADH1) on an existing plasmid in CEB, plasmid pUTK422 was made and transformed, along with the existing pUTK404 (which contains the genes to produce the substrate for the bioluminescent reaction) into the yeast, creating the aryl hydrocarbon-sensing bioluminescent Saccharomyces cerevisiae BLYAHR. Objectives ·Curing S. cerevisiae YCM3 of its colorimetric plasmid (pTXRE5-Z). · Construct the constitutive strain by transforming S. cerevisiae YCM3 with pUTK401 and pUTK404 ·Construct the dioxin-responsive strain by transforming the cured version of YCM3 with pUTK404 and pUTK422 (Bioluminescent Yeast Aryl Hydrocarbon Screen – BLYAhS is created) Test of clones for light production. Six clones were tested for their ability to produce a background level of bioluminescence. Several clones produced higher bioluminescence than the background, indicating that if they are tested with a dioxin there is a good likelihood that they will produce more light, in a dose-responsive fashion. 422404 AhR/ARNT Nucleus Aryl Hydrocarbon Receptor The dioxin-responsive bioluminescence resulting from the transformation of S. cerevisiae YCM3 with pUTK404 and pUTK422. The plasmid pUTK422 was actually the result of using PCR amplification and ligation of the aryl hydrocarbon response elements (XREs) from humans in between two promoters (GPD and ADH1) on an existing plasmid in CEB. This plasmid contains luxAB as well as 5 xenobiotic (also known as aryl hydrocarbon or dioxin) response elements. It produces luciferase, which is the enzyme that performs the bioluminescence reaction. pUTK404 contains the luxCDE from Photorhabdus luminescens and the frp gene from a Vibrio species. The luxCDE genes produce the aldehyde substrate for the bioluminescence reaction. Luciferase, when it's produced either constitutively or in response to a dioxin, breaks down the aldehyde substrate and produces light, quantitatively. The frp gene is also essential as it functions to recycle FMN (flavin mononucleotide) which is a substrate for the reaction. Transforming pUTK422 and pUTK404 into cured S. cerevisiae YCM3 results in a bioreporter that produces bioluminescence in proportion to the dioxin concentration. As a dioxin comes into the cell, it binds to the aryl hydrocarbon receptor, forming a complex. This complex sits on the XREs located in pUTK422 and unwinds the hairpins. This opens up the site for transcription, resulting in luciferase produced from the luxA and luxB genes. This luciferase will then break down the aldehyde that is produced from the luxCDE genes located on pUTK404 and produce light. S. cerevisiae YCM3 was cured of the colorimetric plasmid (pTXRE5-Z). Every day, thirty-two colonies of S. cerevisiae YCM3 were plated onto YPD media and placed overnight in a 28°C incubator so that there was no selective pressure to keep the pTXRE5-Z. The selective marker on pTXRE5-Z is TRP1, so once a week, the thirty-two colonies were also plated onto YMM (+LEU, +URA, -TRP) to monitor the presence of the colorimetric plasmid. This process was repeated for forty-eight rounds until several colonies did not show growth on YMM (+LEU, +URA, -TRP), meaning those colonies had lost pTXRE5-Z. Meanwhile the bioluminesce plasmids pUTK422, pUTK 401, and pUTK404 were isolated. To do so, the plasmids were miniprepped from frozen E. coli clones stored in the CEB freezer. After the plasmids were miniprepped, genes of interest on the resulting plasmids were PCR amplified to confirm these plasmids contained the lux genes. The next step was to form the constitutive bioluminescent S. cerevisiae YCM3 + pUTK 401 + pUTK404 and the dioxin-responsive bioluminescent bioreporter S. cerevisiae YCM3 +pUTK422+ pUTK404 by transforming these plasmids into S. cerevisiae YCM3 that had been cured of the colorimetric plasmid, pTXRE5-Z. This was done using a heat shock method using the Todd Reynolds Lab Transformation Protocol. After the protocol, colonies were tested for plasmid presence. PCR amplification confirmed the presence of pUTK 404 and pUTK 422 in S. cerevisiae YCM3 and thus, the formation of the dioxin-responsive strain. Attempts to construct the constitutive bioreporter, however, are still underway. Methods S. cerevisiae YCM3 has been cured of the colorimetric plasmid, pTXRE5-Z. The dioxin-responsive strain, Bioluminescent Yeast Aryl Hydrocarbon Screen, has been constructed through transformation of pUTK404 and pUTK422 into cured S. cerevisiae YCM3. Attempts to construct the constitutive bioreporter are still underway. The Center for Environmental Biotechnology (CEB) has a history of creating bioluminescent yeast bioreporters that they have successfully used on numerous chemicals to determine if they are estrogenic, androgenic, or toxic (Sanseverino et al. 2009). They have also used these bioreporters on environmental samples (Bergamasco et al. 2011, Jardim et al. 2012). The existing bioreporters are called Saccharomyces cerevisiae BLYES (estrogen-sensing), BLYAS (androgen- sensing), and BLYR (toxicity-sensing). Even though yeast are considered lower eukaryotes, each of these bioreporters contain human proteins and therefore can be considered as a first screen to determine if chemicals will interact (and possibly interfere) with human systems. Through this project, a bioluminescent yeast sensor for the detection of dioxins and aryl hydrocarbons was constructed. Introduction Plasmids used during construction of the bioreporters. To form the constitutive stain, pUTK401 and pUTK404 must be transformed into S. cerevisiae YCM3. To form the dioxin-responsive strain, BLYAhS, pUTK404 and pUTK422 must be transformed into S. cerevisiae YCM3, which contains the human aryl hydrocarbon receptor on its genome. In combination with luxCDABE (on the two plasmids), a dioxin-sensing bioreporter is generated. References 1.A.M.D. Bergamasco, M. Eldridge, J. Sanseverino, F.F. Sodré, C.C. Montagner, I.C. Pescara, W.F. Jardim, G.A. Umbuzeiro (2011) Bioluminescent yeast estrogen assay (BLYES) as a sensitive tool to monitor surface and drinking water for estrogenicity. J Environ Monit 13:3288-3293 2.W.F. Jardim, C.C. Montagner, I.C. Pescara, G.A. Umbuzeiro, A.M.D. Bergamasco, M.L. Eldridge, F. Fabriz Sodré (2012) An integrated approach to evaluate emerging contaminants in drinking water. Sep Purif Technol 84:3-8 3.J. Sanseverino, M.L. Eldridge, A.C. Layton, J.P. Easter, J. Yarbrough, T.W. Schultz, G.S. Sayler (2009) Screening of potentially hormonally active chemicals using bioluminescent yeast bioreporters. Toxicol Sci 107:122-134