Team:TU-Delft/overview

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.

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. Upon detection, the yeast cells will will go in growth arrest and prepare for mating. This mating response can be seen in the form of a morphological change, called shmoo formation.

The image below links to the page explaining more about yeast and why we decided to use it!

Subparts

Receptor

I'd appreciate your input!

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. To make the smelling device to detect tuberculosis, our yeast cells need a Methyl nicotinate- (the scent associated with tuberculosis) receptor. Unfortunately a methyl nicotinate receptor isn't characterized as such yet and we chose a very related ligand receptor: niacin. This receptor, due to it's chimeric properties is transported to the same place as the pheromone receptor and will use the same pathway. In the end our goal is to see whether we can change this receptor through directed mutagenesis to let it smell methyl nicotinate.

To view the plasmids we have made and the accompanying experiments you can click the image below.

At least it puts out

When the cell detects a smell, niacin in this case, we do not want the cell growth to stop, so we deleted the far1-gene, which causes growth arrest. Upon detecting this niacin molecule, we would like to see more than a mating response, the shmoo. For this reason, we added a GFP-output which is promoted by the mating response inducible promoter, FUS1.

Click the image to learn more about our GFP-output!