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| - | {{Template:Tuebingen2012navigation}} | + | {{:Team:Tuebingen/Template/Tuebingen}} |
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| - | *<span style="color:red">This is our provisional page; the final one is still in work. </span>
| + | == The Project == |
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| - | | + | * [[Team:Tuebingen/ProjectOverview|Overview]] <br /> A short introduction including an animation film. |
| - | == '''Overview''' ==
| + | * [[Team:Tuebingen/ProjectQuestions|Questions]] <br /> Every project should start with important questions, here are ours. |
| - | 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.
| + | * [[Team:Tuebingen/ProjectMechanism|Mechanism]] <br /> An in-depth explanation of our genetically engineered system. |
| - | | + | * [[Team:Tuebingen/ProjectImplementation|Implementation]] <br /> Assembly and plasmid construction to implement the system. |
| - | == '''Motivation''' ==
| + | * [[Team:Tuebingen/Parts|Submitted Parts]] <br /> All of our submitted Parts. |
| - | Why do we want to establish a mechanism for steroid measurement?
| + | * [[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 | + | |
| - | * 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 | + | |
| - | *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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| - | == '''Implementation''' ==
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| - | Our System will consist out of 6 parts on 3 different plasmids. Each pair will consist out of a promotor and a protein.
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| - | [[File:Part1.png|thumb|right|Part 1]] | + | |
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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 | + | |
| - | *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 | + | |
| - | *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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