Team:TU-Delft/Project/part2

Abstract

The FUS1 promoter can serve as qualitative reporter for the response to a GPCR activation signal in S. cerevisae. In this section synthesis method and characterization of the FUS1-EGFP biobrick is added. Characterization by using the native pathway by adding alpha pheromone resulted in response when induced with an initial concentration of 2 μM alpha pheromone. No signal increase is observed when altering the alpha pheromone concentration. The promoter leakiness in comparison to response is 4%/73% when induced with 20 μM alpha pheromone. The obtained results can serve as a model for further GPCR research. Finetuning of the experimental method is needed to be able to show response to lower concentrations and possibilities for qualitative response.

Introduction

Yeast FUS1 reporting system

The signal output of a yeast cell with an active receptor is made possible by the FUS1 promoter. The promoter of FUS1 links the MAP kinase pathway to the expression of a chosen protein by having Ste12 inducing the FUS1 response on specific sites of FUS1 (For info about promoters: [http://rulai.cshl.edu/SCPD/ S. cerevisae database]). By using the native receptor Ste2-Ste3 of yeast cells, the original MAPK inducing cascade is used to test FUS1-protein. Main questions are: What is the sensitivity of the FUS1 reporter and does the FUS1 reporter give a quantitative response? To answer this question, YEGFP (Yeast Enhanced GFP) is attached behind the FUS1 promoter to be able to see qualitative and quantitative response in time by using fluorometry measurement techniques. Wildtype yeast strains and Δfar1 (dfar1) yeast strains are used to investigate influence of the original mating response initiated by the gene far1.

Yeast G protein-coupled receptors

In this project we choose to work with the budding yeast Saccharomyces cerevisiae as a host organism because it utilizes already a GPCR pathway. Furthermore S. cerevisiae has been successfully used for functional expression of GPCR’s [3,4], a lot of genomic tools are available, and it has a fully characterized genome. In S. cerevisiae two GPCR cascades have been identified: a glucose sensing pathway and a mating pathway [5]. There are two sexes of yeast cells, MATa and MATα. Whenever pheromones (small peptides) of the opposite sex are bound to the specific G-protein coupled receptors (Ste2 p or Ste3p), the MAP kinase cascade is turned on, leading to induction of mating genes such as FUS1 and growth arrest mediated by the FAR1 promoter. This mating response can be seen in the form of a morphological change, called shmoo formation. In figure 1 an overview of the olfactory sensing pathway can be seen, adapted from Versele et. al S. cerevisiae is shown.

Overview of pheromone and glucose signaling in S. cerevisiae. Figure adapted from Versele et al.