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Team:TU-Delft/overview
From 2012.igem.org
Aim
The goal of this year’s iGEM project is to develop a microbial-based system for the detection of odors, chemicals in gaseous phase. Therefore we will make use of the similarities between the signal transduction cascades of the G-protein coupled receptors (GPCRs) in mammalian cells and the pheromone response pathway in yeast. We aim to functional express mammalian olfactory receptors - GPCRs that bind odorant ligands - in the budding yeast Saccharomyces cerevisiae. By coupling this to a functional reporter it can be used as a novel biosensor for odorant screening. We characterized three mammalian olfactory receptors: one niacin receptor an two isoamyl acetate (bana smell) receptors.
yeast
Yeast, we choose you!
For this project, we will use S. cerevisiae as a host organism because it utilizes already a GPCR pathway: the mating pathway.
Sex response of S. cerevisiae
Yeast genders are called 'a' and 'α', and both genders extract pheromones called 'a'- and 'α'-pheromones. The 'a'-yeasts are able to detect the 'α'-pheromones, and so the other way around. Once the pheromone receptors detects pheromones of another gender, the G-alpha subunit comes to action, dissociating from the GPCR complex. This protein starts a signal leading to growth arrest and to a mating response, of which the morphology is called a shmoo.
The image links to the page explaining more about yeast and why we decided to use it!
Subparts
Receptor
To make the smelling device to detect the ligands that we choose (tubercolosis smell and bananasmell) our yeast cells need to express the corresponding receptors. The receptors, due the chimeric properties that we gave them, are transported to the same place as the pheromone receptor and will use the same pathway.
To view the biobricks we have made and the accompanying experiments you can click the image below.
Reporter
Upon detecting the ligand molecule, we would like to see more than a shmoo mating response. For this reason, we added a EGFP-output which is promoted by the mating response inducible promoter, FUS1.
Click the image to learn more about our EGFP-output!
Olfactory system
By combining the olfactory receptor and the FUS1pr-EGFP reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the underlying cascade is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter.When the cell detects a ligand we do not want the cell to stop growing, so we deleted the FAR1 gene, which causes growth arrest.
