Team:Tuebingen/Project/Overwiew

From 2013.igem.org

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Latest revision as of 20:32, 15 October 2013

Overview

Abstract

Oral contraceptive pills contain progestins as important active components. Excreted progestins are not eliminated from the purified water in sewage treatment plants, thus accumulate in nearby rivers with concentrations up to 15 ng/L. Unfortunately, fish synthesize progestin themselves and use this hormone for the regulation of various processes – contaminant progestins may therefore disrupt these regulation equilibriums and, for example, cause the masculinization of female fish. To this date few quick and cheap measurement methods for environmental progestins are available, hence we decided to devise an easy-to-use yeast-based measurement-system that employs progestin membrane-receptors derived from Danio rerio and Xenopus leavis. These receptors efficiently detect various contaminant progestins due to their low specificity; due to their peripheral localization there is no slowing lag-time thus quick responses are expected. Once progestin binds to the G-protein coupled receptors they start inhibiting the fet3 promotor which in turn regulates a repressor-protein that represses the following promotors (anb1 and suc2). In that way, the receptor-signal is inverted. However, Panb1 (or Psuc2) regulates the downstream reporter-gen luciferase (or lacZ). By using these reporters, we can easily create optical signals that can be analyzed qualitatively without additional equipment or quantified via a spectrometer.

Contraceptive pills usually contain (among other active components) progestin (up to 0.5 mg per pill). After the progestin has made its way through the female body, the hormonal substance is excreted. The progestin carrying sewage water eventually reaches a sewage treatment plant where the sewage water is processed and the resulting purified water ends in a river. Unfortunately, sewage treatment plants cannot eliminate hormonal substances from the water, thus progestin accumulates in nearby rivers where the progestin-contaminated purified water mixes with river-water. Researchers have found mean progestin levels in sewage treatment plants of about 1 ng/L sewage water – in the river-water outside of one particular American sewage treatment plant 15 ng/L progestin was measured. In lab-studies, researchers have found that progestin-concentrations as low as 0.8 ng/L can have an effect on fish.

Fish can synthesize progestin themselves and use this hormone (similar to humans) for the regulation of sexual differentiation – they therefore have some kind of progestin-receptor. It has been shown in lab-studies that the specificity of these receptors is not very high since even very low concentrations of artificial progestins were able to induce responses when binding these receptors. Therefore it is quite possible that progestin-contaminated water can induce such responses under natural conditions as well – plus exaggerated progestin-concentrations might have unexpected effects.

Naturally, progestin is secreted by female fish in order to induce the display of mating behavior in males. While displaying mating behavior males stop foraging, show increased activity, and similar expensive behavioral patterns. Contaminant progestin could induce this behavior at inopportune times thus increase predation risk and generally exhaust males. Additionally, contaminant progestin decreases sperm motility and disturbs spermatogenesis. Also, contaminant progestin disrupts embryonic development and induces masculinization in female fish (which in turn decreases clutch size). All in all, progestin is highly dangerous for aquatic ecosystems and further decreases the chances of survival of small populations.

The effects of progestin and other so called endocrine disruptive substances (EDS) on aquatic life-forms have not been studied in depth until very recently and there are only few methods for measuring the progestin-concentration in water.

This lack of easy and quick measurement systems inspired us to develop a progestin measurement system based on yeast. Our system will employ progestin receptors of Danio rerio and Xenopus laevis as precise detectors and firefly luciferase as an efficient reporter. We hope to at least be able to make qualitative measurements with our system (which will be possible without any additional equipment!) but our ultimate goal are precise quantitative measurements using a simple photometer. Since we have to produce most of the BioBricks we want to use ourselves, we have no data about their performance. As a result, we cannot easily predict successful BioBrick-combinations – as a way to counter possible erroneous predictions we have chosen a modular approach that enables us to simply replace inefficient parts with more fitting constructs in order to maximize overall performance.

In order to achieve quick and precise hormone-detection we utilize G-protein coupled membrane-receptors (Progesterin- and AdipoQ-Receptor family) which have relatively low progestin-specificity thus might be able to respond to various (artificial) contaminant progestins. We decided to favor membrane-receptors over intracellular receptors because intracellular receptors tend to have longer lag-phases thus slow down the overall speed of our system. The first promotor in our system will be the promotor of the FET3 gene which is specifically inhibited by the membrane receptors. Once progestin binds to the receptor the inhibition is initiated thus downstream genes cannot be read by polymerase. Pfet3 regulates a repressor gene (either mig1 or rox1) which is terminated by adh1. The repressor mig1 / rox1 inhibits the next promotor in our system: either Psuc2 or Panb1 (respectively)– in effect the repressor thus functions as an inverter for the receptor-signal: as long as no progestin is detected by the receptors the repressor represses transcription of the promotor Panb1 / Psuc2 - once progestin binds to the receptors the repressor is not expressed anymore thus downstream genes of Panb1 / Psuc2 are transcribed. Panb1 / Psuc2 regulates the reporter gene (preferably luciferase (LUC) – we develop lacZ as a substitute – we call our system “modular” for a reason! EDIT: lacZ was dismissed as a reporter after multiple PCRs did not have any results.) which is terminated by Tadh1.

Exemplary cellular overview of our measurement system (inside a yeast cell) when there is no contaminant progestin in the yeast's medium. MIG1 / ROX1 represses Psuc2 / Panb1 thus there is no expression of luc.

Exemplary cellular overview of our measurement system (inside a yeast cell) when there is contaminant progestin in the yeast's medium. mPR Dr represses Pfet3 thus there is no expression of mig1 / rox1. Psuc2 / Panb1 is not repressed thus (via expression of luc) LUC catalyzes its bioluminescence reaction.

As said before, we hope to construct a cost-efficient, easy-to-use, and quick measurement system for contaminant progestin in rivers or lakes. We will use the remaining time until October to maximize the usability of our system in order that only minimum staff-training is required before reliable quantitative measurements can be conducted. Our aim is the compilation of progestin contamination data along rivers so that specific solutions for each pollution can be found.

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@@ Line 17: / Line 17: @@
 <div id="actualAbstract">
-<h1 id="actualAbstractTitle">Abstract</h1>
+<h3>Abstract</h3>
-<p style="padding:0.5em" align="justify">Oral contraceptive pills contain progestins as important active components. Excreted progestins are not eliminated from the purified water in sewage treatment plants thus accumulate in nearby rivers with concentrations up to 15 ng/L. Unfortunately fish synthesize progestin themselves and use this hormone for the regulation of various processes – contaminant progestins may therefore disrupt these regulation equilibriums and for example cause the masculinization of female fish. To this date few quick and cheap measurement methods for environmental progestins are available hence we decided to devise an easy-to-use yeast-based measurement-system that employs progestin membrane-receptors derived from <i>Danio rerio</i> and <i>Xenopus leavis</i>. These receptors efficiently detect various contaminant progestins due to their low specificity; due to their peripheral localization there is no slowing lag-time thus quick responses are expected. Once progestin binds to the G-protein coupled receptors they start inhibiting the fet3 promotor which in turn regulates a repressor-protein that represses the following promotors (anb1 and suc2). In that way the receptor-signal is inverted. However, Panb1 (or Psuc2) regulates the downstream reporter-gen luciferase (or lacZ). By using these reporters we can easily create optical signals that can be analyzed qualitatively without additional equipment or quantified via a spectrometer.</p>
+<p style="padding:0.5em" align="justify">Oral contraceptive pills contain progestins as important active components. Excreted progestins are not eliminated from the purified water in sewage treatment plants, thus accumulate in nearby rivers with concentrations up to 15 ng/L. Unfortunately, fish synthesize progestin themselves and use this hormone for the regulation of various processes – contaminant progestins may therefore disrupt these regulation equilibriums and, for example, cause the masculinization of female fish. To this date few quick and cheap measurement methods for environmental progestins are available, hence we decided to devise an easy-to-use yeast-based measurement-system that employs progestin membrane-receptors derived from <i>Danio rerio</i> and <i>Xenopus leavis</i>. These receptors efficiently detect various contaminant progestins due to their low specificity; due to their peripheral localization there is no slowing lag-time thus quick responses are expected. Once progestin binds to the G-protein coupled receptors they start inhibiting the fet3 promotor which in turn regulates a repressor-protein that represses the following promotors (anb1 and suc2). In that way, the receptor-signal is inverted. However, Panb1 (or Psuc2) regulates the downstream reporter-gen luciferase (or lacZ). By using these reporters, we can easily create optical signals that can be analyzed qualitatively without additional equipment or quantified via a spectrometer.</p>
 </div>
-<p style="margin-top:2em">Contraceptive pills usually contain (among other active components) progestin (about 0.1 mg per pill). After the progestin has made its way through the female body the hormonal substance is excreted. The progestin carrying sewage water eventually reaches a sewage treatment plant where the sewage water is processed and the resulting purified water ends in a river. Unfortunately sewage treatment plants cannot eliminate hormonal substances from the water thus progestin accumulates in nearby rivers where the progestin-contaminated purified water mixes with river-water. Researchers have found mean progestin levels in sewage treatment plants of about 1 ng/L sewage water – in the river-water outside of one particular American sewage treatment plant 15 ng/L progestin was measured. In lab-studies researchers have found that progestin-concentrations as low as 0.8 ng/L can have an effect on fish.</p>
+<p style="margin-top:2em">Contraceptive pills usually contain (among other active components) progestin (up to 0.5 mg per pill). After the progestin has made its way through the female body, the hormonal substance is excreted. The progestin carrying sewage water eventually reaches a sewage treatment plant where the sewage water is processed and the resulting purified water ends in a river. Unfortunately, sewage treatment plants cannot eliminate hormonal substances from the water, thus progestin accumulates in nearby rivers where the progestin-contaminated purified water mixes with river-water. Researchers have found mean progestin levels in sewage treatment plants of about 1 ng/L sewage water – in the river-water outside of one particular American sewage treatment plant 15 ng/L progestin was measured. In lab-studies, researchers have found that progestin-concentrations as low as 0.8 ng/L can have an effect on fish.</p>
 <p>Fish can synthesize progestin themselves and use this hormone (similar to humans) for the regulation of sexual differentiation – they therefore have some kind of progestin-receptor. It has been shown in lab-studies that the specificity of these receptors is not very high since even very low concentrations of artificial progestins were able to induce responses when binding these receptors. Therefore it is quite possible that progestin-contaminated water can induce such responses under natural conditions as well – plus exaggerated progestin-concentrations might have unexpected effects.</p>
-<p>Naturally progestin is secreted by female fish in order to induce the display of mating behavior in males. While displaying mating behavior males stop foraging, show increased activity, and similar expensive behavioral patterns. Contaminant progestin could induce this behavior at inopportune times thus increase predation risk and generally exhaust males. Additionally, contaminant progestin decreases sperm motility and disturbs spermatogenesis. Also, contaminant progestin disrupts embryonic development and induces masculinization in female fish (which in turn decreases clutch size). All in all progestin is highly dangerous for aquatic ecosystems and further decreases the chances of survival of small populations.</p>
+<p>Naturally, progestin is secreted by female fish in order to induce the display of mating behavior in males. While displaying mating behavior males stop foraging, show increased activity, and similar expensive behavioral patterns. Contaminant progestin could induce this behavior at inopportune times thus increase predation risk and generally exhaust males. Additionally, contaminant progestin decreases sperm motility and disturbs spermatogenesis. Also, contaminant progestin disrupts embryonic development and induces masculinization in female fish (which in turn decreases clutch size). All in all, progestin is highly dangerous for aquatic ecosystems and further decreases the chances of survival of small populations.</p>
 <p>The effects of progestin and other so called endocrine disruptive substances (EDS) on aquatic life-forms have not been studied in depth until very recently and there are only few methods for measuring the progestin-concentration in water.</p>
-<p>This lack of easy and quick measurement systems inspired us to develop a progestin measurement system based on yeast. Our system will employ <a href="https://2013.igem.org/Team:Tuebingen/Project/Receptor">progestin receptors</a> of <i>Danio rerio</i> and <i>Xenopus laevis</i> as precise detectors and <a href="https://2013.igem.org/Team:Tuebingen/Project/Reporter">firefly luciferase as an efficient reporter</a>. We hope to at least be able to make qualitative measurements with our system (which will be possible without any additional equipment!) but our ultimate goal are precise quantitative measurements using a simple photometer. Since we have to produce most of the BioBricks we want to use ourselves we have no data about their performance. As a result we cannot easily predict successful BioBrick-combinations – as a way to counter possible erroneous predictions we have chosen a modular approach that enables us to simply replace inefficient parts with more fitting constructs in order to maximize overall performance.</p>
+<p>This lack of easy and quick measurement systems inspired us to develop a progestin measurement system based on yeast. Our system will employ <a href="https://2013.igem.org/Team:Tuebingen/Project/Receptor">progestin receptors</a> of <i>Danio rerio</i> and <i>Xenopus laevis</i> as precise detectors and <a href="https://2013.igem.org/Team:Tuebingen/Project/Reporter">firefly luciferase as an efficient reporter</a>. We hope to at least be able to make qualitative measurements with our system (which will be possible without any additional equipment!) but our ultimate goal are precise quantitative measurements using a simple photometer. Since we have to produce most of the BioBricks we want to use ourselves, we have no data about their performance. As a result, we cannot easily predict successful BioBrick-combinations – as a way to counter possible erroneous predictions we have chosen a modular approach that enables us to simply replace inefficient parts with more fitting constructs in order to maximize overall performance.</p>
+<a name="cellular_mechanism"> </a>
 <p>In order to achieve quick and precise hormone-detection we utilize G-protein coupled membrane-receptors (Progesterin- and AdipoQ-Receptor family) which have relatively low progestin-specificity thus might be able to respond to various (artificial) contaminant progestins. We decided to favor membrane-receptors over intracellular receptors because intracellular receptors tend to have longer lag-phases thus slow down the overall speed of our system.
 The first promotor in our system will be the promotor of the FET3 gene which is specifically inhibited by the <a href="https://2013.igem.org/Team:Tuebingen/Project/Receptor">membrane receptors</a>. Once progestin binds to the receptor the inhibition is initiated thus downstream genes cannot be read by polymerase. Pfet3 regulates a repressor gene (either mig1 or rox1) which is terminated by adh1. The repressor mig1 / rox1 inhibits the next promotor in our system: either Psuc2 or Panb1 (respectively)– in effect the repressor thus functions as an <a href="https://2013.igem.org/Team:Tuebingen/Project/Inverter">inverter</a> for the receptor-signal: as long as no progestin is detected by the receptors the repressor represses transcription of the promotor Panb1 / Psuc2 - once progestin binds to the receptors the repressor is not expressed anymore thus downstream genes of Panb1 / Psuc2 are transcribed. Panb1 / Psuc2 regulates the <a href="https://2013.igem.org/Team:Tuebingen/Project/Reporter">reporter</a> gene (preferably luciferase (LUC) – we develop lacZ as a substitute – we call our system “modular” for a reason! <i>EDIT: lacZ was dismissed as a reporter after multiple PCRs did not have any results.</i>) which is terminated by Tadh1.</p>
-<div style="padding: 10px; height: 420px; width: 690px; background-color: #CCCCCC; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/c/c7/TueComplete_nocontamination.PNG" style="position: relative; width: 680px; margin: 5px 5px 10px 5px;"><b>Fig. 1</b>: Cellular overview of our measurement system (inside a yeast cell) when there is no progestin contamination in the yeast's medium.</div>
+<div style="padding: 10px; height: auto; width: 690px; background-color: #CCCCCC; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/2/2c/TueCellularOverview_withoutProgestin.PNG" style="position: relative; width: 680px; margin: 5px 5px 10px 5px;">Exemplary cellular overview of our measurement system (inside a yeast cell) when there is no contaminant progestin in the yeast's medium. MIG1 / ROX1 represses Psuc2 / Panb1 thus there is no expression of luc.</div>
+<p>&nbsp;</p>
+<div style="padding: 10px; height: auto; width: 690px; background-color: #CCCCCC; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/e/ee/TueCellularOverview_withProgestin.PNG" style="position: relative; width: 680px; margin: 5px 5px 10px 5px;">Exemplary cellular overview of our measurement system (inside a yeast cell) when there is contaminant progestin in the yeast's medium. mPR Dr represses Pfet3 thus there is no expression of mig1 / rox1. Psuc2 / Panb1 is not repressed thus (via expression of luc) LUC catalyzes its bioluminescence reaction.</div>
-<p>As said before we hope to construct a cost-efficient, easy-to-use, and quick measurement system for contaminant progestin in rivers or lakes. We will use the remaining time until October to maximize the usability of our system in order that only minimum staff-training is required before reliable quantitative measurements can be conducted. Our aim is the compilation of progestin contamination data along rivers so that specific solutions for each pollution can be found.</p>
+<p>As said before, we hope to construct a cost-efficient, easy-to-use, and quick measurement system for contaminant progestin in rivers or lakes. We will use the remaining time until October to maximize the usability of our system in order that only minimum staff-training is required before reliable quantitative measurements can be conducted. Our aim is the compilation of progestin contamination data along rivers so that specific solutions for each pollution can be found.</p>
 <p>&nbsp;</p>
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