Team:Tuebingen/Project

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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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* [[Team:Tuebingen/ProjectQuestions|Questions]] <br /> Every project should start with important questions, here are ours.
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Our general aim is to establish a simple synthetic organisim which will
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* [[Team:Tuebingen/ProjectMechanism|Mechanism]] <br /> An in-depth explanation of our genetically engineered system.
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be capable to measure the influence of endocrine disruptors on the
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* [[Team:Tuebingen/ProjectImplementation|Implementation]] <br /> Assembly and plasmid construction to implement the system.
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natural balance of sexual determination in all kind of vertebrates. The
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* [[Team:Tuebingen/Parts|Submitted Parts]] <br /> All of our submitted Parts.
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measurement itself will be cost-efficient, environment-friendly and
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* [[Team:Tuebingen/References|References]] <br /> Scientific literature used to design our project.
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sensitive.
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Naturally occuring iron receptors of the PAQR family are found to
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repress the fet3 promotor on high level of extra-cellular iron.
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According to Smith et al. human mPR expressed in yeast induced the same
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signal when binding to progesterone. Relying on this results we will
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express various mPRs of Danio rerio and Xenopus laevis in yeast to
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measure endocrine substances that influence fish and amphibians.
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We will transform the negative signal (fet3 repression) into a positive
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signal by regulating a repressor (rox1 or mig1) with Pfet3.  This
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repressor will in turn regulate the expression of the reporter gene
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(firefly luciferase or beta-galactosidase) and allow quantitative
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measurement.
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== Motivation ==
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''Why do we want to establish a mechanism for steroid measurement?''
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Steroid hormones, especially estrogens, occur in all vertebrates and
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play a crucial role in sexual differentiation. In recent times the
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pollution of waters with these hormones has become an increasing problem
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for the aquatic fauna.
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Particularly waters functionalized by humans or adjacent to human
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settlements, e.g. in areas with agricultural use, show increased
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concentrations of estrogen.
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Scientific studies based on ''Danio rerio'' showed that the consequences
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are devastating.
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High concentrations of 17α-ethinylestradiol, a hormone in most
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birth-control pills, affected the sex differentiation of ''Danio rerio''
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leading to development of ovotestis or complete feminization (Andersenc,
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2002).
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Intersex-fish have been reported in UK rivers since 1978 downstream of
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an sewage treatment plant.
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We believe that a first step in finding a solution to this environmental
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problem is an accurate and reliable method to quantify steroid
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concentrations.
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== Occurring Questions ==
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On our way designing the major pathway to express a specific reporter
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gene to demonstrate the presence of steroid hormones, we had and still
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have to deal with several questions concering the choice of BioBricks,
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genes and vectors to construct a firm method to determine "pollution" by
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steroids. As a conclusion, we have to meet two major requirements for
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our system:
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* It should be as cost-efficient as possible for easy and regular
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application
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* It should be resistant to yeast's own metabolism (Not be disturbed by
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unexpected occuring expression).
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At first we had to find an appropriate receptor to "grab" steroid
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hormones in efluents.  This should fulfill the following requirements:
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* It should only be responsible to substrates we wish to detect, so the
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results of the test will not be falsified.
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* It has to be easiliy integrated into yeast's cell membrane.
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* Its nucleic acid sequence should not be too long so we can put it onto
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a plasmid vector.
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We chose the membrane progesterone receptor of zebra fish (''Danio
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rerio'') and of African clawed frog (''Xenophus laevis''). We focused on
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these receptors, since they are easy to duplicate and interact with a
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broad bandwith of sex-determining hormones.
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The next step was to select an appropriate organism to express these
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receptors. After some research, we could narrow our options down to two
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organisms:
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* Escherichia coli
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* Saccharomyces cerevisiae
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Finally we decided for yeast, since it has been done more research with
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it according to our prefered receptors. In addition yeast is an
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eukaryote making it more easier to integrate mPRs into their cell
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membrane.
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== '''Mechanism''' ==
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[[File:Igem_2.3.jpg|thumb|right|Planned mechanism]]
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Naturally occuring iron receptors of the PAQR family are found to block
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FET3 promotor on high level of extra cellular iron. According to
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[http://www.ncbi.nlm.nih.gov/pubmed/18603275 Smith JL et al] expressed
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human mPR in yeast induced the same signal ligating to estrogen. Relying
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on this results we will try to express various mPRs of ''Danio rerio'',
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''Xanophus laevis'' in yeast which we find better fitting to measure
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endocrine substances that influence fish.
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We will transform the negative signal of FET3 by letting it control an
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inhibitor (ROX1 and MIG1). This inhibitor will regulate the promotor of
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our reporter gene. For the promoter of our reporter gene we chose ANB1
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and SUC2, because these are targets of ROX1 and MIG1. By negating the
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negative signal of FET3 we hope to obtain a positive signal sensitive
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enough to measure low concentrations of different endocrine substances.
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=== Receptors ===
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Membrane bound, 7-Transmembranereceptor (C-terminus inside,N-terminus
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outside), PAQR family (progesterone adiponectin Q receptor), Hly-III
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superfamily G-Protein coupled: activation of inhibitori Gi units:
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reduced adenylyl-cyclase activity. 
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=== Inhibitors and their targets ===
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An appropriate inhibitor/promotor combination is a crucial step in our
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pathway and should be selected wisely.
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Finally we chose FET3 as our promoter and both ROX1 and MIG1 as
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inhibitors. It is very likely, that they don't seem to repress any gene
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expression that are crucial for yeast.
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=== Reporter genes ===
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The last station of our signaling pathway should be a reporter gene
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which amplifies our initial signal to allow a quantitative  measurement.
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The enzyme '''luciferase''' fulfills these conditions and is our
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candidate of choice.
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== Implementation ==
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=== Promoter ===
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[[File:PRS313_Padh1_Danio_Tadh1.png|promoter1]]
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[[File:PRS313_Padh1_Xenopus_Tadh1.png|promoter2]]
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=== Inhibitor ===
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[[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor1]]
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[[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor2]]
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=== Reporter ===
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[[File:PRS316_Panb1_lacZ_Tadh1.png|reporter1]]
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[[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter2]]
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[[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter3]]
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== Measurement ==
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The measurement itself should be limited to an optical one.  With this
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idea in mind, we decided for reporter genes like lacZ and luciferase,
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because these produce signals which can simply be quantified by optical
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measurement methods.
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== References ==
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* [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
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* [2] Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment
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<!--
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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
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rapid nongenomic signaling of zebrafish membrane progestin receptors
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alpha and beta in transfected cells
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* Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in
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Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in
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Hefezellen. (Detection of doping relevant anabolic steroids in horse
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urin and blood plasma using yeast reporter gene assays)
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* “Assault on the male”, BBC 1996. Video report
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http://www.youtube.com/watch?v=LkxIJJI37bQ
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* Dr. Volker Scheil, The impact of potential environmental stressors on
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early development and cellular and biochemical biomarkers in fish,
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Main research: Fish embryotoxicity, histopathology and stress protein
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(hsp 70) responses.
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* Harris et al. - 2011 - The consequences of feminization in breeding
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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
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endocrine disruption in the aquatic environment from known knowns to
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unknown unknowns (and many things in between)
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* Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout
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(Salvelinus namaycush) exposed to environmentally relevant
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concentrations of the potent estrogen ethynylestradiol (EE2) in a
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whole lake exposure experiment
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* Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An
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environmentally relevant concentration of estrogen induces arrest of
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male gonad development in zebrafish, Danio rerio. Environmental
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toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from
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http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)
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* Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., &
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Segner, H. (2007). Concentration- and time-dependent effects of the
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synthetic estrogen, 17alpha-ethinylestradiol, on reproductive
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capabilities of the zebrafish, Danio rerio. Journal of toxicology and
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environmental health. Part A, 70(9), 768-79.
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doi:10.1080/15287390701236470
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* Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe,
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D., Schäfers, C., et al. (2003). Identification of
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endocrine-disrupting effects in aquatic vertebrates and invertebrates:
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report from the European IDEA project. Ecotoxicology and environmental
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safety, 54(3), 302-14. Retrieved from
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http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)
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* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and
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rapid nongenomic signaling of zebrafish membrane progestin receptors
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alpha and beta in transfected cells
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* Thomas et al. - 2007 - Steroid and G protein binding characteristics
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of the seatrout and human progestin membrane receptor alpha subtypes
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and their evolutionary origins
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* Thomas - 2008 - Characteristics of membrane progestin receptor alpha
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(mPR ) and progesterone membrane receptor component 1 (PGMRC1) and
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their roles in mediating rapid progestin actions
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* Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang,
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Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons  - 2008 -
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Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms
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their ability to function as membrane progesterone receptors
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-->
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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.