Team:EPFL/Overview

Espress'EAU - Synthetic Biology

Introduction

The transcription factors, Msn2p and Msn4p, are master regulators of the stress response pathway in yeast.1 They will start activating downstream genes upon stresses, such as carbon source starvation, heat shock and severe osmotic or oxidative stresses. Hsp12 is one of these target genes and codes for a small heat shock protein. Its function is to maintain membrane organization during stress conditions. Yap1p is a transcriptional regulator that controls the response to oxidative stress.2 It controls a number of target genes such as TRX2, GSH1, YCF1 and GLR1. TRX2 codes for a protein that is part of the thioredoxin system which protects cells against oxidative and reductive stress. GSH1 codes for the enzyme that catalyzes the first step in glutathione (GSH) biosynthesis. YCF1 codes for an ABC transporter that transports glutathione from the cytosol into vacuoles and is required for vacuole fusion. GLR1 codes for a cytosolic and mitochondrial glutathione oxidoreductase.3 We chose a selection of genes regulated by either MSN2/4 or Yap1p (Figure 1) and created transcriptional units that would allow the cell to become a biosensor for various environmental stresses.

Figure 1

Figure 1: MSN2 and MSN4 are the master regulators of carbon source starvation, heat shock and severe osmotic or oxidative stresses. YAP1 is the master regulator of oxidative stress. After receiving a stress signal, the master regulators will be activated and translocalized towards the nucleus where they will up-regulate their target genes1,2.

Deletion Strains

To find out about the growth behavior of yeast strains in which the master regulators are knocked out we performed plate reader experiments to measure growth curves.

Figure 1

Figure 2: MSN2 and wild-type strains OD600 measurements across time, with and without Bentazon added. The concentration of Bentazon was 1mg/L in a solution of 10% Methanol.

Reporter Strains

Figure 1

We integrated the reporter protein mScarlet-I under the control of five different stress response promoters in the genome of S.Cerevisiae. Fluorescent microscopy data shows that the protein is expressed at different levels in the five strains that we created. We tested the strains on the plate reader and found that the expression of mScarlet-I can be induced by the pesticide Metolachlor solved in Methanol.

References

  1. Martínez-Pastor, M. T. et al. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15, 2227–2235 (1996).
  2. Coleman, S. T., Epping, E. A., Steggerda, S. M. & Moye-Rowley, W. S. Yap1p Activates Gene Transcription in an Oxidant-Specific Fashion. Molecular and Cellular Biology 19, 8302–8313 (1999).
  3. Saccharomyces Genome Database | SGD [Internet]. [cited 2020 Oct 26]. Available from: https://www.yeastgenome.org/
  4. Gietz, R. D. & Schiestl, R. H. Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols 2, 35–37 (2007).

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