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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<br/>
 
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</>
 
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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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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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<br/>
 
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<h2>references</h2>  <br/>
 
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<h2>Methods</h2>  <br/>
 
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<h3>Expression vectors</h3>
 
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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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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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<h3>yeast transformation and growth</h3>
 
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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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<h3>Fluorometer experiment</h3>
 
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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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<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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<h2>Results</h2>  <br/>
 
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<h2>Content</h2>
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<a href="#P10"> Introduction</a><br>
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<a href="#P9">Parts </a><br>
 +
<a href="#P8">Results </a><br>
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<a href="#P7"> Conclusions</a><br>
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<a href="#P6"> Recommendations </a><br>
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<a href="#P5">References</a><br>
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<a name="P10"> <br><h2>Introduction</h2> </a>
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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. 
 +
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>
 +
<img src="http://igem.org/wiki/images/a/a2/Reportersuperchill.png" width="40%" height="35%"
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<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6>
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<a name="P9"> <h2>Parts</h2> </a>
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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/>
 +
<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="http://igem.org/wiki/images/d/dd/Reporter.png" align="middle" width="500"><br/>
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<a name="P8"> <h2>Results</h2><a name="A4"> </a>
 +
<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>
 +
<h6>Outcome</h6>
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<img src="http://igem.org/wiki/images/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>
 +
<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>
 +
<h6>Setup</h6>
 +
<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.
 +
<h6>Outcome</h6>
 +
<img src="http://igem.org/wiki/images/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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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/>
 +
<br>
 +
<h3>Variability of the receptor: single cell microscopy</h3>
 +
<h6>Description</h6>
 +
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="http://2012.igem.org/Team:TU-Delft/informationtheory"> Information processing module </a>.<br>
 +
An example is shown of the process imaging which is done:<br><br>
 +
<img src="http://2012.igem.org/wiki/images/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>
 +
 +
<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>
 +
<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/>
 +
[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/>
 +
[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

Menu

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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)