Team:TU-Delft/part2

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<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6>
 
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<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6>
<h2>Parts</h2>  
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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/>  
<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/>  

Revision as of 00:32, 27 September 2012

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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. The MAP kinase cascade is responsible for the activation of STE12 which acts as the transcription factor for the FUS1 gene. If a reporter gene as EGFP is coupled to the FUS1 promoter the FUS1 promoter can be characterized. The main questions are: What is the sensitivity of the FUS1-EGFP reporter and does the FUS1-EGFP reporter give a quantitative response? To answer this questions, YEGFP (Yeast Enhanced GFP) is cloned behind the FUS1 promoter to be able to see qualitative and quantitative response in time by using fluorometry measurement techniques.

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 Growth (OD600) and Fluorescence intensity of S. cerevisiae transformed with FUS1pr-EGFP. A. Growth curve of alpha pheromone induced cells. B. Intensity curve of alpha induced cells. C. Intensity/Growth of alpha pheromone induced cells.

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).

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

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. 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).

Conclusions

The rapid decrease in OD600 can be explained by a maximum amount of yeast cells before piling begins. 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 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 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)