Team:TU-Delft/part2

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<h2>Introduction</h2>  
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<p>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/)</p>
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<h2>Content</h2>  
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<br/>
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<a href="#P10"> Introduction</a><br>
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</>
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<a href="#P9">Parts </a><br>
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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?
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<a href="#P8">Results </a><br>
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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.)</p>
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<a href="#P7"> Conclusions</a><br>
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<br/>
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<a href="#P6"> Recommendations </a><br>
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<h2>references</h2> <br/>
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<a href="#P5">References</a><br>
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<h2>Methods</h2> <br/>
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<h3>Expression vectors</h3>
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<a name="P10"> <br><h2>Introduction</h2> </a>
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<p>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)
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<p>There are two sexes of yeast cells, MATa and MATα. Whenever pheromones of the opposite sex are bound to the specific G-protein coupled receptors the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i>. If a reporter gene as EGFP is coupled to the <i>FUS1</i> promoter the <i>FUS1</i> MAP kinase cascade can be characterized. 
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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>
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The main questions are: What is the sensitivity of the <i>FUS1-EGFP</i> reporter, does the <i>FUS1-EGFP</i> reporter give a quantitative response, hoe long does it take before you can see a response? To answer this questions, <i>YEGFP</i> (Yeast Enhanced GFP) is cloned behind the <i>FUS1</i> promoter and the native yeast receptors are induced by alpha pheromones. With fluorometry measurement techniques we are able to see qualitative and quantitative response in time. </p>
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<h3>yeast transformation and growth</h3>
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<img src="https://static.igem.org/mediawiki/igem.org/a/a2/Reportersuperchill.png" width="40%" height="35%"
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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>
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<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6>
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<h3>Fluorometer experiment</h3>
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<a name="P9"> <h2>Parts</h2> </a>
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<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>
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<p>The <i>FUS1pr-EGFP</i> construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a single nucleotide deletion in EGFP gene and therefore we cloned another EGFP behind the FUS1 promoter. The EGFP that is used is obtained from the <a href="http://www.addgene.org/14196/">pAG416GPD-ccdB-EGFP</a> plasmid (kindly provided by Harmen van Rossum from Delft University of Technology). The plasmid construct was obtained by restriction and ligation in the pRSII415 expression vector. The following biobrick was created:<br/>  
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775004 " target="_blank">BBa_K775004</a><br/></p><br/>
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<td> <img src="https://static.igem.org/mediawiki/igem.org/d/dd/Reporter.png" align="middle" width="500"><br/>
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<a name="P8"> <h2>Results</h2><a name="A4"> </a>
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<h3>Fluorometer experiment</h3><a name="A2"> </a>
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<h6>Setup</h6>
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<p>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 (excitation 485/20, emission 590/35) is measured every 1.40 minutes.</p>
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<h6>Outcome</h6>
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<img src="https://static.igem.org/mediawiki/igem.org/f/f4/FUS1fluorometer.png" border="0" width="80%" height="45%"><br>
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<h6>Fluorometer data of <i>S. cerevisiae</i> transformed with <i>FUS1pr-EGFP</i></h6>
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<p>A GFP response is expected in yeasts transformed with <i>FUS1pr-EGFP</i> when induced with alpha pheromone. The growth curve, EGFP intensity curves and EGFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=3 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. For the 200 nM the peak level is at t=2. 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).</p>
<h3>Flow cytometry experiment</h3>
<h3>Flow cytometry experiment</h3>
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<p>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. </p>
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<h6>Setup</h6>
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<h2>Results</h2>  <br/>
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<p>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.
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<h3>Fluorometer experiment</h3>  <br/>
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<h6>Outcome</h6>
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<p>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).</p> <br/>  
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<img src="https://static.igem.org/mediawiki/igem.org/0/07/FUSGFPFACS.jpg" border="0" width="90%" height="60%"><br><h6>The upper part shows Side Scattering versus fluorescence intensity. Side Scattering is an indication of the morphological structure of the cells. The lower part shows a histogram of the fluorescence intensity distribution.</h6>
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<p>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. </p>
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In the figure a signal intensity shift can be observed from the I=3*10^2 towards I=2*10^3 between the WT strain induced with alpha pheromone and the <i>FUS1pr-EGFP</i> strain induced with alpha pheromone (2nd and 3rd column). This indicates that yeast cells react to the alpha pheromone with a fluorescent signal. If we look at the non induced <i>FUS1pr-EGFP</i> strain (first column) we can see that a small region correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the <i>FUSpr1-EGFP</i> (leakiness). </p><br/>
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<h3>Flow cytometry experiment</h3>  <br/>
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<br>
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<p>Flow cytometri on <i>S. Cerevisae</i> 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). <p> <br/>
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<h3>Variability of the receptor: single cell microscopy</h3>
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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.
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<h6>Description</h6>
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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>
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With single cell microscopy we monitor the behaviour of the cellular response towards a ligand in time. For this complex process of monitoring a separate page is introduced: <a href="https://2012.igem.org/Team:TU-Delft/informationtheory"> Information processing module </a>.<br>
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<h2>Discussion and conclusions</h2>  <br/>
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An example is shown of the process imaging which is done:<br><br>
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<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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<img src="https://static.igem.org/mediawiki/2012/9/90/BF_cell_indexing.png" width="500" align="center"/><br><h6> image of Saccharomyces cerevisae, masked with tracking numbers which follow the cells over time</h6><br>
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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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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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 +
<a name="P7"> <h2>Conclusions</h2> </a>
 +
<p> An increase in EGFP expression can be seen when yeast cells are induced with concentrations of 200 nM 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2-3 hours after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependent of the type of vector used and whether plasmid or chromosomal integration is chosen.</p><br/>
 +
<a name="P16"> <h2>Recommendations</h2> </a>
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<p>Further investigation should be done on fine-tuning the promoter affinity by varying the sequence of the <i>STE12p</i> docking sites and experimental conditions by estimating influencing parameters during an experiment. This includes:  estimating alpha pheromone degradation rate/cell.</p><br/>
 +
<br/>
 +
<a name="P5"> <h2>references</h2> </a>
 +
<h6>[1] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001<br/>
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[2]Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/>
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[3] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001)</h6>
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Latest revision as of 03:56, 27 October 2012

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Content

Introduction
Parts
Results
Conclusions
Recommendations
References

Introduction

There are two sexes of yeast cells, MATa and MATα. Whenever pheromones of the opposite sex are bound to the specific G-protein coupled receptors the MAP kinase cascade is turned on, leading to induction of mating genes such as FUS1. If a reporter gene as EGFP is coupled to the FUS1 promoter the FUS1 MAP kinase cascade can be characterized. The main questions are: What is the sensitivity of the FUS1-EGFP reporter, does the FUS1-EGFP reporter give a quantitative response, hoe long does it take before you can see a response? To answer this questions, YEGFP (Yeast Enhanced GFP) is cloned behind the FUS1 promoter and the native yeast receptors are induced by alpha pheromones. With fluorometry measurement techniques we are able to see qualitative and quantitative response in time.

Schematic overview of the FUS1-EGFP reporter

Parts

The FUS1pr-EGFP construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a single nucleotide 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). The plasmid construct was obtained by restriction and ligation in the pRSII415 expression vector. The following biobrick was created:
BBa_K775004



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 (excitation 485/20, emission 590/35) is measured every 1.40 minutes.

Outcome

Fluorometer data of S. cerevisiae transformed with FUS1pr-EGFP

A GFP response is expected in yeasts transformed with FUS1pr-EGFP when induced with alpha pheromone. The growth curve, EGFP intensity curves and EGFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=3 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. For the 200 nM the peak level is at t=2. 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).

Flow cytometry experiment

Setup

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.

Outcome

The upper part shows Side Scattering versus fluorescence intensity. Side Scattering is an indication of the morphological structure of the cells. The lower part shows a histogram of the fluorescence intensity distribution.
In the figure a signal intensity shift can be observed from the I=3*10^2 towards I=2*10^3 between the WT strain induced with alpha pheromone and the FUS1pr-EGFP strain induced with alpha pheromone (2nd and 3rd column). This indicates that yeast cells react to the alpha pheromone with a fluorescent signal. If we look at the non induced FUS1pr-EGFP strain (first column) we can see that a small region correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the FUSpr1-EGFP (leakiness).



Variability of the receptor: single cell microscopy

Description
With single cell microscopy we monitor the behaviour of the cellular response towards a ligand in time. For this complex process of monitoring a separate page is introduced: Information processing module .
An example is shown of the process imaging which is done:


image of Saccharomyces cerevisae, masked with tracking numbers which follow the cells over time

Conclusions

An increase in EGFP expression can be seen when yeast cells are induced with concentrations of 200 nM 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2-3 hours after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependent of the type of vector used and whether plasmid or chromosomal integration is chosen.


Recommendations

Further investigation should be done on fine-tuning the promoter affinity by varying the sequence of the STE12p docking sites and experimental conditions by estimating influencing parameters during an experiment. This includes: estimating alpha pheromone degradation rate/cell.



references

[1] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001
[2]Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)
[3] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001)