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From 2012.igem.org

Overview

Our general aim is to establish a simple synthetic biological which allows us to measure the concentration of substances that influence the natural balance of sexual determination in all kind of vertebrates. The measurement itself should be cost-efficient, environment-friendly and as precise as possible. Therefore we want to clone the membrane progestin receptor (mPR) of the zebrafish (Danio rerio) and salmon (Salmo salar) into yeast (Saccharomyces cerevisiae). We decided in favour of this receptor because it is capable to interact with a high bandwidth of sexual determining substances (e.g. estrogene). To acquire an authentic result, the receptor is linked to a reporter gene, which allows us to evaluate the concentration by a simple optical measurement.

Motivation

Why do we want to establish a mechanism for steroid measurement?

Steroid hormones, especially estrogens, occur in all vertebrates and play a crucial role in sexual differentiation. In recent times the pollution of waters with these hormones has become an increasing problem for the aquatic fauna. Particularly waters functionalized by humans or adjacent to human settlements, for example in areas with agricultural use, show increased estrogen-concentrations.

Scientific studies based on Danio rerio showed that the consequences are devastating. High concentrations of 17α-ethinylestradiol, a hormone in most birth-control pills, affected the sex differentiation of Danio rerio leading to development of ovotestis or complete feminization (Andersenc, 2002). Intersex-fish have been reported in UK rivers since 1978 downstream of an sewage treatment plant.

We believe that a first step in finding a solution to this environmental problem is an accurate and reliable method to quantify steroids which is easy to use and limited to an optical measurement.

Occurring Questions

On our way designing the major pathway to express a specific reporter gene to demonstrate the presence of steroid hormones, we had and still have to deal with several questions concering the choice of BioBricks, genes and vectors to construct a firm method to determine "pollution" by steroid hormones. As a conclusion, we have to meet two major requirements for our system:

• It should be as cost-efficient as possible for easy and regular application
• It should be resistant to yeast's own metabolism. (Not be disturbed by unexpected occuring expression)

At first we had to find an appropriate receptor to detect steroid hormones.

Mechanism

Naturally occuring iron receptors of the PAQR family are found to block FET3 promotor on high level of extra cellular iron. According to [http://www.ncbi.nlm.nih.gov/pubmed/18603275 Smith JL et al] expressed human mPR in yeast induced the same signal ligating to estrogen. Relying on this results we will try to express various mPRs of Danio rerio, Salmo salar in yeast which we find better fitting to measure endocrine substances that influence fish.

We will transform the negative signal of FET3 by letting it control an inhibitor. This inhibitor will regulate the promotor of our reporter gene. By negating the negative signal of FET3 we hope to obtain a positive signal sensitive enough to measure low concentrations of different endocrine substances.

Receptors

Membrane bound, 7-Transmembranereceptor (C-terminus inside,N-terminus outside), PAQR family (progesterone adiponectin Q receptor), Hly-III superfamily G-Protein coupled: activation of inhibitori Gi units: reduced adenylyl-cyclase activity.

Sequence mPR alpha (Zebrafish): [http://www.ncbi.nlm.nih.gov/nuccore/AY149121.1 NCBI],

Sequence mPR beta (Zebrafish): [http://www.ncbi.nlm.nih.gov/nuccore/AY149120.1 NCBI]

Homologe receptors of mPR Alpha Danio rerio:

• Goldfisch, Carassius auratus
• Katzenwels, Ictalurus punctatus
• Hundszungen, Cynoglossus semilaevis
• Flunder, Paralichthys lethostigma
• Meerforelle, Cynoscion nebulosus
• Buntbarsch, Oreochromis niloticus
• Umberfisch, Micropogonias undulatus

Zebrafrisch (Danio rerio) In Tübingen:

• Tübingen Map of the [http://wwwmap.tuebingen.mpg.de/ Zebrafish Genome]
• [http://www.mnf.uni-tuebingen.de/fachbereiche/biologie/institute/evolutionecology/lehrbereiche/physiologische-oekologie-der-tiere/staff/volker-scheil.html Dr. Volker Scheil], The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.

Inhibitor and promotor

An appropriate inhibitor/promotor combination is a crucial step in our pathway and should be selected wisely.

Reporter gene

The last station of our signaling pathway should be a reporter gene which amplifies our initial signal to allow a quantitative measurement.

The enzyme luciferase fulfills these conditions and is possible candidate for our mechanism.

Implementation

Our System will consist out of 6 parts on 3 different plasmids. Each pair will consist out of a promotor and a protein.

Plasmid #1: mPR

+

Plasmid #2: Inhibitor

+

Plasmid #3: Reporter

+

Measurement

The measurement itself should be limited to an optical one and people with lesser qualifications should be able to run it.

References

• [1] Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors
• [2] Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment
• Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish
• Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
• Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in Hefezellen. (Detection of doping relevant anabolic steroids in horse urin and blood plasma using yeast reporter gene assays)
• “Assault on the male”, BBC 1996. Video report http://www.youtube.com/watch?v=LkxIJJI37bQ
• Dr. Volker Scheil, The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.
• Harris et al. - 2011 - The consequences of feminization in breeding groups of wild fish
• Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish
• Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on endocrine disruption in the aquatic environment from known knowns to unknown unknowns (and many things in between)
• Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout (Salvelinus namaycush) exposed to environmentally relevant concentrations of the potent estrogen ethynylestradiol (EE2) in a whole lake exposure experiment
• Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio. Environmental toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)
• Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., & Segner, H. (2007). Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio. Journal of toxicology and environmental health. Part A, 70(9), 768-79. doi:10.1080/15287390701236470
• Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe, D., Schäfers, C., et al. (2003). Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project. Ecotoxicology and environmental safety, 54(3), 302-14. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)
• Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
• Thomas et al. - 2007 - Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins
• Thomas - 2008 - Characteristics of membrane progestin receptor alpha (mPR ) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions
• Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors