From 2012.igem.org

Latest revision as of 09:27, 26 September 2012

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 has to be easiliy integrated into yeast's cell membrane.
• It should only be responsible to substrates we wish to detect, so the results of the test will not be falsified.
• 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 zebrafish (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.