Team:TU-Delft/part2

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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). </p>
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). </p>
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<h2>Results</h2>  <br/>
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<h2>Results</h2>
<h3>Fluorometer experiment</h3>
<h3>Fluorometer experiment</h3>
<h6>Setup</h6>
<h6>Setup</h6>
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<p>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.
<p>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 %. </p><br/>
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 %. </p><br/>
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<h2>Discussion and conclusions</h2>   
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<h2>Conclusions</h2>   
<p>The rapid decrease in OD600 can be explained by a maximum amount of yeast cells before piling begins. Also fluctuations during the measurement in the room could be the cause of a significant  absorption drop.
<p>The rapid decrease in OD600 can be explained by a maximum amount of yeast cells before piling begins. Also fluctuations during the measurement in the room could be the cause of a significant  absorption drop.
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An increase in GFP expression can be seen when yeast cells are induced with 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2.40 after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependant of the type of vector used and whether plasmid or chromosomal integration is chosen.
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An increase in EGFP expression can be seen when yeast cells are induced with 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2.40 after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependant of the type of vector used and whether plasmid or chromosomal integration is chosen.</p>
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Further investigation should be fine tuning the promoter affinity and experimental conditions by estimating influencing parameters during an experiment. This includes:  estimating alpha pheromone degradation rate/cell.</p>
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<h2>Recommendations</h2>
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<p>Further investigation should be fine tuning the promoter affinity and experimental conditions by estimating influencing parameters during an experiment. This includes:  estimating alpha pheromone degradation rate/cell.</p>
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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


Parts

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).

Results

Fluorometer experiment

Setup

Exponential growth phase cells were put into a 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.

A GFP response is expected in yeasts transformed with FUS1pr-EGFP when induced with alpha pheromone. 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

When cells were in exponential phase they were induced with alpha pheromone. Cells where then measured with a Cytek FACScan. Graphs were analyzed with Flowjo. In the figure 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 FUS1pr-EGFP strain correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the FUSpr1-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 %.


Conclusions

The rapid decrease in OD600 can be explained by a maximum amount of yeast cells before piling begins. Also fluctuations during the measurement in the room could be the cause of a significant absorption drop. An increase in EGFP expression can be seen when yeast cells are induced with 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2.40 after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependant of the type of vector used and whether plasmid or chromosomal integration is chosen.

Recommendations

Further investigation should be fine tuning the promoter affinity and experimental conditions by estimating influencing parameters during an experiment. This includes: estimating alpha pheromone degradation rate/cell.