Team:TU-Delft/part2

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Introduction

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/)

By using the native receptor Ste2-Ste3 of yeast cells, the original MAPK inducing cascade is used to test FUS1pr-protein. Main questions are: What is the sensitivity of the FUS1pr reporter and does the FUS1pr 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 far1Δ::KANMX (dfar1) yeast strains are used to investigate influence of the original mating response initiated by the gene FAR1.)

references

Methods

Expression vectors

All plasmid manipulations were performed in E. coli strain DH5α Top10 cells from Invitrogen (?). After selection, a single colony was used to isolate the plasmid (with miniprep) for yeast transformation. The plasmid construct for the receptor expression was obtained by restriction and ligation in the pRS415-II expression vector (called FUS1-EGFP) The FUS1-EGFP construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a deletion in EGFP gene and therefore we cloned another EGFP behind the Fus1 promoter. The EGFP that is used is obtained from the pAG416GPD-ccdB-EGFP plasmid (kindly provided by Harmen van Rossum from Delft University of Technology).

yeast transformation and growth

The S. cerevisiae S288C strain Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 and S. cerevisiae S288C strain Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δfar1 were transformed with the FUS1-EGFP expression vectors. Transformed cells were plated on 2% agar synthetic dropout media according to Verduyn et all. [Verduyn et all.] without LEU. The presence of plasmid in transformed cells was verified using PCR on purified plasmids (using zymolyase, see protocols). Primers used annealed on the EGFP gene and pRS415II backbone.

Fluorometer experiment

Yeast strains were picked from -80 C stock and inoculated overnight. Yeast is grown until the afternoon, cells OD600 is evened to approximately 0.1, put into 96 well plate and cells are induced with alpha pheromone. Cells are mixed and OD600 signal and GFP signal is measured every 1.40 minutes.

Flow cytometry experiment

A frozen stock is grown overnight on 30 °C and diluted until an OD600 of approximately 0.05 was measured. Cells were grown in DO –Leucine media. Cells where centrifuged (10 min 4000 RPM) and media was refreshed. Cells were grown for seven hours and centrifuged again. Pellet size is estimated and evened. Media was refreshed again. After 2.5 hours of growth (cells in exponential phase) cells were induced with ligand. Cells where then measured with a Cytek FACScan. Graphs were analyzed with Flowjo.

Results

Fluorometer experiment

A GFP response is expected in yeasts transformed with FUS1pr-EGFP when induced with alpha pheromone. A fluorometer experiment is set up. The growth curve, GFP intensity curves and GFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=2.40 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. After this, the intensity decreases to normal again. Interesting is that there can be found an almost linear correlation between GFP intensity and growth for lower concentrations (seen as lines in the lower graph).

Deviations in cell density can be due to mixing of the 96 wells plate. Yeast cells tend to go to the bottom and with higher cell densities they cluster and form a layer. The fluorometer mixes by shaking a perfect round circle causing the yeast cells to pile up into a wavy line (seen at the end of the experiment), which causes less absorption per cell density and thus a drop of OD600.

Flow cytometry experiment

Flow cytometri on S. Cerevisae FUS1pr-EGFP has been performed. Results can be seen in figure 4. Here a signal intensity shift can be observed from the I=3*102 towards I=2*103. This indicates that yeast cells react to the alpha pheromone with fluorescent signal. Also a small region in the non induced FUS1-EGFP strain correlates with higher intensity (thus GFP expression). This is probably the signal noise of the FUS1-EGFP (leakiness).

The subsets give an indication on cell intensity distribution by setting a limit to the amount of tolerated deviation (a cloud) and counting the amount of cells in the cloud relative to the total amount. When the data is divided into subsets (figure 4) which correlate with intensity clouds, the size of the non-induced high intensity subset is 4 % of the total amount where the size of the induced high intensity subset is 73 %.

Discussion and conclusions