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From 2012.igem.org

Project Overview

Our general aim is to establish a simple synthetic organisim which will be capable to measure the influence of endocrine disruptors on the natural balance of sexual determination in all kind of vertebrates. The measurement itself will be cost-efficient, environment-friendly and 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 (Andersen, 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.