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Team:TU-Delft/part2
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<p>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. </p> | <p>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. </p> | ||
<h3>Fluorometer experiment</h3> | <h3>Fluorometer experiment</h3> | ||
| + | <p>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.</p> | ||
| + | <h3>Flow cytometry experiment</h3> | ||
| + | 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. | ||
Revision as of 16:19, 25 September 2012
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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.
