Who are we?
In the following months we will be developing a yeast-based biosensor for the sleeping hormone melatonin. Melatonin regulates the circadian rhythm and plays an important role in depression, Alzheimer’s and Parkinson’s disease. Therefore, we want to facilitate melatonin measurement by building a less expensive biosensor which can be used without special laboratory equipment.
Our approach is to genetically modify Saccharomyces cerevisiae by integrating a highly specific human melatonin receptor into the cells. This will allow us to detect melatonin from a saliva sample without previous purification. To get a signal from the melatonin binding, we will use two different signalling systems:
Using a nuclear receptor and a reporter gene:
The membrane permeability for melatonin is high. This permits us to use the nuclear retinoid z receptor (RZR) which is directly regulating gene expression. As the genes regulated by RZR do not exist in S. cerevisiae, we plan to create a chimeric receptor consisting of the RZR and the recognition sequence of the human estrogen receptor alpha (ERα). When melatonin is bound, the modified receptor can bind to the estrogen receptor responsive element (ERE) and as a consequence regulate expression of firefly luciferase reporter genes. The signal intensity of luciferase is dose-dependent in the presence of ligands.
Enzyme-Fragment Complementation Assay:
When melatonin binds to the G protein-coupled receptor MT1, β-arrestins can be recruited. We are going to use this mechanism by designing two fusion-proteins: on the one hand the MT1 receptor will be fused to a fragment of an enzyme, on the other hand, the complementing enzyme fragment will be fused to the β-arrestin. Thus, receptor activation leads to β-arrestin recruitment and therefore to the formation of an active enzyme.