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-	__TOC__	+	== The Project ==
-	== Overview ==	+	* [[Team:Tuebingen/ProjectOverview|Overview]] <br /> A short introduction including an animation film.
-		+	* [[Team:Tuebingen/ProjectQuestions|Questions]] <br /> Every project should start with important questions, here are ours.
-	Our general aim is to establish a simple synthetic organisim which will	+	* [[Team:Tuebingen/ProjectMechanism|Mechanism]] <br /> An in-depth explanation of our genetically engineered system.
-	be capable to measure the influence of endocrine disruptors on the	+	* [[Team:Tuebingen/ProjectImplementation|Implementation]] <br /> Assembly and plasmid construction to implement the system.
-	natural balance of sexual determination in all kind of vertebrates. The	+	* [[Team:Tuebingen/Parts|Submitted Parts]] <br /> All of our submitted Parts.
-	measurement itself will be cost-efficient, environment-friendly and	+	* [[Team:Tuebingen/References|References]] <br /> Scientific literature used to design our project.
-	sensitive.	+
-		+
-	Naturally occuring iron receptors of the PAQR family are found to	+
-	repress the fet3 promotor on high level of extra-cellular iron.	+
-	According to Smith et al. human mPR expressed in yeast induced the same	+
-	signal when binding to progesterone. Relying on this results we will	+
-	express various mPRs of Danio rerio and Xenopus laevis in yeast to	+
-	measure endocrine substances that influence fish and amphibians.	+
-		+
-	We will transform the negative signal (fet3 repression) into a positive	+
-	signal by regulating a repressor (rox1 or mig1) with Pfet3.  This	+
-	repressor will in turn regulate the expression of the reporter gene	+
-	(firefly luciferase or beta-galactosidase) and allow quantitative	+
-	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, e.g. in areas with agricultural use, show increased	+
-	concentrations of estrogen.	+
-		+
-	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 steroid	+
-	concentrations.	+
-		+
-	== 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	+
-	steroids. 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 (no disturbance by unexpected expression).	+
-		+
-	At first we had to find an appropriate receptor to bind steroid	+
-	hormones in efluents. It should fulfill the following requirements:	+
-		+
-	* It should only be responsible to substrates we wish to detect, so the results of the test will not be falsified.	+
-	* It has to be easiliy integrated into yeast's cell membrane.	+
-	* Its nucleic acid sequence should not be too long so we can put it onto a plasmid vector.	+
-		+
-	We chose the membrane progesterone receptor of the zebra fish (''Danio rerio'') and the African clawed frog (''Xenopus laevis''). We focused on	+
-	these receptors, since they are easy to duplicate and interact with a	+
-	broad bandwith of sex-determining hormones.	+
-		+
-	The next step was to select an appropriate organism to express these	+
-	receptors. After some research, we could narrow our options down to two	+
-	organisms:	+
-		+
-	* ''Escherichia coli''	+
-	* ''Saccharomyces cerevisiae''	+
-		+
-	Finally we decided for yeast, since it has been done more research with	+
-	it according to our prefered receptors. In addition yeast is an	+
-	eukaryote making it more easier to integrate mPRs into their cell	+
-	membrane.	+
-		+
-	== '''Mechanism''' ==	+
-		+
-	[[File:Igem_2.3.jpg|thumb|right|Planned mechanism]]	+
-		+
-	[[File:MechanismScheme.png|thumb|Mechanism]]	+
-		+
-	Naturally occuring iron receptors of the PAQR family are found to repress	+
-	fet3 promotor on high levesl of extra-cellular iron. According to	+
-	[http://www.ncbi.nlm.nih.gov/pubmed/18603275 J Smith et al.]	+
-	human mPR expressed in yeast induced the same signal binding to estrogen. Relying	+
-	on this results we will try to express various mPRs of ''Danio rerio'',	+
-	''Xanophus laevis'' in yeast which we find fitting to measure	+
-	endocrine substances that influence fish.	+
-		+
-	We will transform the negative signal (fet3 repression) into a positive signal by regulating a repressor (rox1 or mig1) with Pfet3. This repressor will in turn regulate the expression of the reporter gene (firefly luciferase or beta-galactosidase) and allow quantitative measurement.	+
-		+
-	=== Receptors ===	+
-		+
-	Membrane progesterone receptors are membrane bound, seven-transmembrane receptors and belong to the PAQR family (progesterone adiponectin Q receptor). These G-protein coupled receptors activate inhibitory Gi units.	+
-		+
-	=== Inhibitors and their targets ===	+
-		+
-	An appropriate inhibitor/promotor combination is a crucial step in our	+
-	pathway and should be selected wisely.	+
-		+
-	Finally we chose FET3 as our promoter and both ROX1 and MIG1 as	+
-	inhibitors. It is very likely, that they don't seem to repress any gene	+
-	expression that are crucial for yeast.	+
-		+
-	=== Reporter genes ===	+
-		+
-	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 our	+
-	candidate of choice.	+
-		+
-	== Implementation ==	+
-		+
-		+
-	=== Promoter ===	+
-	[[File:PRS313_Padh1_Danio_Tadh1.png|promoter1]]	+
-	[[File:PRS313_Padh1_Xenopus_Tadh1.png|promoter2]]	+
-	=== Inhibitor ===	+
-	[[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor1]]	+
-	[[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor2]]	+
-	=== Reporter ===	+
-	[[File:PRS316_Panb1_lacZ_Tadh1.png|reporter1]]	+
-	[[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter2]]	+
-	[[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter3]]	+
-		+
-	== Measurement ==	+
-		+
-	The measurement itself should be limited to an optical one.  With this	+
-	idea in mind, we decided for reporter genes like lacZ and luciferase,	+
-	because these produce signals which can simply be quantified by optical	+
-	measurement methods.	+
-		+
-	== 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	+
-		+
-	-->	+

---

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.