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Team:Tuebingen/Playground
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Revision as of 16:07, 30 May 2012
Contents |
Overview
Our general aim is to establish a simple synthetic biological which allows us to measure the concentration of substances that influence the natural balance of sexual determination in all kind of vertebrates. The measurement itself should be cost-efficient, environment-friendly and as precise as possible. Therefore we want to clone the membrane progestin receptor (mPR) of the zebrafish (Danio rerio) and salmon (Salmo salar) into yeast (Saccharomyces cerevisiae). We decided in favour of this receptor because it is capable to interact with a high bandwidth of sexual determining substances (e.g. estrogene). To acquire an authentic result, the receptor is linked to a reporter gene, which allows us to evaluate the concentration by a simple optical 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, for example in areas with agricultural use, show increased estrogen-concentrations.
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 steroids which is easy to use and limited to an optical measurement.
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 steroid hormones. 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. (Not be disturbed by unexpected occuring expression)
At first we had to find an appropriate receptor to detect steroid hormones.
Mechanism
Naturally occuring iron receptors of the PAQR family are found to block FET3 promotor on high level of extra cellular iron. According to Smith JL et al expressed human mPR in yeast induced the same signal ligating to estrogen. Relying on this results we will try to express various mPRs of Danio rerio, Salmo salar in yeast which we find better fitting to measure endocrine substances that influence fish.
We will transform the negative signal of FET3 by letting it control an inhibitor. This inhibitor will regulate the promotor of our reporter gene. By negating the negative signal of FET3 we hope to obtain a positive signal sensitive enough to measure low concentrations of different endocrine substances.
Receptors
Membrane bound, 7-Transmembranereceptor (C-terminus inside,N-terminus outside), PAQR family (progesterone adiponectin Q receptor), Hly-III superfamily G-Protein coupled: activation of inhibitori Gi units: reduced adenylyl-cyclase activity.
Sequence mPR alpha (Zebrafish): NCBI,
Sequence mPR beta (Zebrafish): NCBI
Homologe receptors of mPR Alpha Danio rerio:
- Goldfisch, Carassius auratus
- Katzenwels, Ictalurus punctatus
- Hundszungen, Cynoglossus semilaevis
- Flunder, Paralichthys lethostigma
- Meerforelle, Cynoscion nebulosus
- Buntbarsch, Oreochromis niloticus
- Umberfisch, Micropogonias undulatus
Zebrafrisch (Danio rerio) In Tübingen:
- Tübingen Map of the Zebrafish Genome
- 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.
Inhibitor and promotor
An appropriate inhibitor/promotor combination is a crucial step in our pathway and should be selected wisely.
Reporter gene
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 possible candidate for our mechanism.
Implementation
Our System will consist out of 6 parts on 3 different plasmids. Each pair will consist out of a promotor and a protein.
Plasmid #1: mPR
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Plasmid #2: Inhibitor
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Plasmid #3: Reporter
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Measurement
The measurement itself should be limited to an optical one and people with lesser qualifications should be able to run it.
