Team:Tuebingen/Project

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*<span style="color:red">This is our provisional page; the final one is still in work. </span>
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== The Project ==
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== '''Overview''' ==
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* [[Team:Tuebingen/ProjectOverview|Overview]] <br /> A short introduction including an animation film.
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Our general aim is to establish a simple mechanism which allows us to measure an estrogen concentration in a solution as precisely as possible. Therefore we want to clone the membrane progestin receptor (mPR) of the zebrafish (''Danio rerio'') in yeast (''saccharomyces cerevisiae'') and link it to a reporter gene, which allows a quantitative  measurement.
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* [[Team:Tuebingen/ProjectQuestions|Questions]] <br /> Every project should start with important questions, here are ours.
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* [[Team:Tuebingen/ProjectMechanism|Mechanism]] <br /> An in-depth explanation of our genetically engineered system.
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== '''Motivation''' ==
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* [[Team:Tuebingen/ProjectImplementation|Implementation]] <br /> Assembly and plasmid construction to implement the system.
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Why do we want to establish a mechanism for steroid measurement?
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* [[Team:Tuebingen/Parts|Submitted Parts]] <br /> All of our submitted Parts.
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* [[Team:Tuebingen/References|References]] <br /> Scientific literature used to design our project.
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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.
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Particularly waters functionalized by humans or adjacent to human settlements, for example in areas with agricultural use, show increased estrogen-concentrations.
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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).
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Intersex-fish have been reported in UK rivers since 1978 downstream of an sewage treatment plant.
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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.
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== ''Occurring Questions'' ==
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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:
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* It should be as cost-efficient as possible for easy and regular application
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* It should be resistant to yeast's own metabolism. (Not be disturbed by unexpected occuring expression)
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At first we had to find an appropriate receptor to detect steroid hormones.
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== '''Mechanism''' ==
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[[File:IGem.jpg]]
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=== '''Receptors''' ===
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Membrangebunden, 7-Transmembranrezeptor (Innen C-Terminus, außen N-Terminus), PAQR-Familie (progesterone adiponectin Q receptor), Hly-III superfamily
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G-Protein gekoppelt: Aktivierung von inhibitorische Gi-Einheiten: Verringerung der Aktivität der Adenylyl-Cyclase 
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G Proteins (Protein Data Bank) Artikel zu G-Proteinen - Struktur und Funktionsweise, schöne Bilder
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Sequenz mPR alpha (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149121.1 NCBI],
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Sequenz mPR beta (Zebrafisch):  [http://www.ncbi.nlm.nih.gov/nuccore/AY149120.1 NCBI]
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Homologe zum mPR Alpha Danio rerio:
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*Goldfisch, Carassius auratus
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*Katzenwels, Ictalurus punctatus
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*Hundszungen, Cynoglossus semilaevis
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*Flunder, Paralichthys lethostigma
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*Meerforelle, Cynoscion nebulosus
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*Buntbarsch, Oreochromis niloticus
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*Umberfisch, Micropogonias undulatus
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Zebrafrisch (Danio rerio)
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In Tübingen:
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*Tübingen Map of the [http://wwwmap.tuebingen.mpg.de/ Zebrafish Genome]
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*[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.
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=== '''Inhibitor and promotor''' ===
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An appropriate inhibitor/promotor combination is a crucial step in our pathway and should be selected wisely.
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=== '''Reporter gene''' ===
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The last station of our signaling pathway should be a reporter gene which amplifies our initial signal to allow a quantitative  measurement.
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The enzyme '''luciferase''' fulfills these conditions and is possible candidate for our mechanism.
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== '''Measurement''' ==
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The measurement itself should be limited to an optical one and people with lesser qualifications should be able to run it.
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== '''References''' ==
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*Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment
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*Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish
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*Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
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*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)
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*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
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*“Assault on the male”, BBC 1996. Video report http://www.youtube.com/watch?v=LkxIJJI37bQ
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*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.
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*Harris et al. - 2011 - The consequences of feminization in breeding groups of wild fish
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*Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish
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*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)
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*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
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*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)
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*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
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*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)
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*Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
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*Thomas et al. - 2007 - Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins
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*Thomas - 2008 - Characteristics of membrane progestin receptor alpha (mPR ) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions
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*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
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Latest revision as of 14:46, 18 September 2012



The Project

  • Overview
    A short introduction including an animation film.
  • Questions
    Every project should start with important questions, here are ours.
  • Mechanism
    An in-depth explanation of our genetically engineered system.
  • Implementation
    Assembly and plasmid construction to implement the system.
  • Submitted Parts
    All of our submitted Parts.
  • References
    Scientific literature used to design our project.