Team:TU-Delft/part3

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Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/>
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/>
<h3>Increasing sensitivity</h3>
<h3>Increasing sensitivity</h3>
-
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p>
+
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. At the beginning of this project we designed and ordered the sequence for a alpha subunit that can be integrated into the chromosomal DNA. Also we made a strain with a double knockout: of the alpha subunit gene and the <i>FAR1</i> gene. In future work the alpha subunit should be expressed and characterized. </p>
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<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/>
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/>
<br/>
<br/>
-
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br>
+
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>dfar1</i>-strain shows less reduction. </br>
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br>
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br>
</p>
</p>
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<img src="http://igem.org/wiki/images/6/66/Groeicurve.jpg" align="middle" width="100%">
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<img src="http://igem.org/wiki/images/1/18/Groeicurve2.jpg" align="middle" width="100%"></br>
-
 
+
<br></br><br>
<h3>Transformations</i></h3>
<h3>Transformations</i></h3>
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output  
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output  
-
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (nicotinic acid reporter). The <i>RI7-ODR10</i> receptor and output  
+
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (niacin reporter). The <i>RI7-ODR10</i> receptor and output  
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output  
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output  
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p>
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p>
-
<br/>
+
</br>
<p>
<p>
-
<h3>Niacin Snifferomyces</h3><br/>
+
<h3>Niacin Snifferomyces</h3>
-
<h6>Setup</h6><br/>
+
<h6>Setup Fluorescence microscopy</h6>
-
In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.
+
Since there is now a fluorescence output we can analyze our Snifferomyces with fluorescent microscopy. The transformed cells were induced with niacin, alpha pheromone and we kept a control that was not induced. After 3,5 hours we viewed the cells with a fluorescence microscope. <br>
-
<h6>Outcome</h6><br/>
+
<h6>Outcome Fluorescence microscopy</h6>
-
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.
+
<img src="http://igem.org/wiki/images/b/b3/Screen-capture-14.jpg" width="500">
 +
<h6>Non induced, niacin induced and alpha pheromone induced Snifferomyces NR1 cells</h6><br>
 +
We analyzed the cells with ImageJ and made histograms of the fluorescencent values. In the graphs we see not induced, niacin induced and alpha pheromone induced Snifferomyces NR1 cells. Here we can see a difference between the niacin induced cells and the non induced cells: there is a shift in fluorescence distribution towards the higher fluorescence for the niacin induced cells. This indicates, as we also observed in the reporter part of the project, that the niacin ligand binds to the receptor. In the third graph we can see that there is a much higher fluorescence value for the cells induced with alpha pheromone.
 +
Further we can see that the non induced cells show a fluorescent value, this is what we already had observed in the reporter part of the project: the <i>FUS1</i> promoter is leaky.</br></br>
 +
 
 +
<h6>Setup Flow cytometry</h6>
 +
<p>In order to test the yeast Snifferomyces NR1 cells with niacin receptor and the receptor inducible GFP reporter we induced the cells with niacin or alpha pheromone and measured the cellular response through flow cytometry, measuring the EGFP signal intensity.</p><br>
 +
<h6>Outcome Flow cytometry</h6>
 +
<p>Flow cytometry data of +NR1 cells induced with niacin or alpha pheromones is shown.</p>
</p>
</p>
-
<img src=”http://2012.igem.org/wiki/images/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png” alt="FC results" width=”350” />
+
<img src="http://2012.igem.org/wiki/images/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png" width="600">
<p>
<p>
-
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:
+
Here we see that addition of receptor ligand doesn’t induce an increased light response in comparison with alpha induced cells. Possible explanations are:
<ul><li>A  too small subset of the yeast receptors is localized in the membrane.</li>
<ul><li>A  too small subset of the yeast receptors is localized in the membrane.</li>
-
<li>The affinity of the receptor with the alpha subunit is too low.</li>
+
<li>The affinity of the receptor with the alpha subunit is too low.</li><br>
-
 
+
-
 
+
<h3>Isoamyl acetate Snifferomyces</h3>
<h3>Isoamyl acetate Snifferomyces</h3>
<h6>Setup</h6>
<h6>Setup</h6>
 +
<p>Since oily undissolved isoamyl acetate can disturb the flow cytomtery experiments a new setup was designed. In this setup we blew air together with gaseous isoamyl acetate trough the yeast culture. In the setup we could measure the offfas CO2 and O2 and we could take samples in time for flow cytometry. The fermenter setup is shown in the figure.</p>
 +
<img src="http://igem.org/wiki/images/b/bb/Screen-capture-15.jpg" width="400">
<h6>Outcome</h6>
<h6>Outcome</h6>
 +
<p>We runned in total four fermenters: two with the Snifferomyces strain and two with a reference strain. In both cases one of the fermenters was blown with isoamyl acetate and air and the other one only with air. We saw that in both the experiments were the isoamyl acetate was blown trough the fermenters the cells stopped growing. After the isoamyl acetate blow was turned off the cells started to grow again. Thus this experiment tells us that both the reference strain and the Snifferomyces strain cells cannot grow with this high amount of isoamyl acetate.</p>
 +
<br> </br></br>
 +
<a name="P7"> <h2>Conclusions</h2> </a>
<a name="P7"> <h2>Conclusions</h2> </a>
-
<p>Four olfactory system biobricks were added to the registry. </p><br/>
+
<p>Four olfactory system biobricks were added to the registry. The light response when adding niacin ligand to the NR1 Snifferomyces cells was seen with single cells (fluorescence microscopy), but didn't lead to statistical significant data in the flow cytometry experiment. For the banana receptor Snifferomyces we observed that cells were high concentrations of isoamyl acetate were blown trough, stopped growing. </p><br/>
 +
<a name="P8"> <h2>Recommendations</h2> </a>
 +
To increase expression and light intensity of the cells in response to a ligand the following conditions can be tested:
 +
<ul><li> Make the receptor expression time as short as possible. Use an inducible promoter or make stocks as soon as possible after transformation </li>
 +
<li> There are indications that succesful expression can be altered by decreasing the temperature during receptor expression. When we tested this the growth rate was slow and therefore the expression time became too long. An inducible promoter can solve this problem too.</li>
 +
<li> The affinity towards the downstream pathway could be increased when the pheromone binding subunit alpha is knocked out (<i>dGPA1</i>) and replaced by a mammalian one (typically <a href="http://www.ncbi.nlm.nih.gov/protein/47271350?report=genbank&log$=prottop&blast_rank=1&RID=8NZM5VY001R">NP_034437.1</a> is used. We didn't have the time to test this hypothesis, further investigation is needed!</li></ul><br/>
 +
 
<a name="P9"><h2>References</h2> </a>
<a name="P9"><h2>References</h2> </a>
<h6>[1] 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)</h6>
<h6>[1] 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)</h6>
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6>
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6>
 +
 +
<a name="P8"> <h2>Recommendation</h2></a>
 +
</div>
</div>
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Latest revision as of 04:00, 27 October 2012

Menu

Receptor

Content

Introduction
Parts
Results
Conclusions
References

Introduction

Yeast with olfactory receptor+reporter=Snifferomyces

By combining the olfactory receptor and the FUS1pr-EGFP reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the FAR1 promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.

Growth arrest

Besides the induction of the FUS1 promoter the cells also go in growth arrest mediated by the FAR1 promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the FAR1 promoter. Therefore we characterized the knockout of the FAR1 promoter.

Increasing sensitivity

To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. At the beginning of this project we designed and ordered the sequence for a alpha subunit that can be integrated into the chromosomal DNA. Also we made a strain with a double knockout: of the alpha subunit gene and the FAR1 gene. In future work the alpha subunit should be expressed and characterized.

Parts

The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:


BBa_K775005
BBa_K775006
BBa_K775007
BBa_K775008

Results

Characterization Far1 Knockout

The FAR1 has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones.

The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the dfar1-strain shows less reduction.
Another remarkable feature is that the Δfar1-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The FAR1 is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2]





Transformations

Yeast strains were successfully transformed with the GPR109A receptor and output BBa_K775005, called NR1 (niacin reporter). The RI7-ODR10 receptor and output BBa_K775008. Olfr154 receptor and output BBa_K775010, called BR1 (banana reporter 1) and RI7-OR1G1 receptor and output BBa_K775011, called BR2 (banana reporter 2).


Niacin Snifferomyces

Setup Fluorescence microscopy
Since there is now a fluorescence output we can analyze our Snifferomyces with fluorescent microscopy. The transformed cells were induced with niacin, alpha pheromone and we kept a control that was not induced. After 3,5 hours we viewed the cells with a fluorescence microscope.
Outcome Fluorescence microscopy
Non induced, niacin induced and alpha pheromone induced Snifferomyces NR1 cells

We analyzed the cells with ImageJ and made histograms of the fluorescencent values. In the graphs we see not induced, niacin induced and alpha pheromone induced Snifferomyces NR1 cells. Here we can see a difference between the niacin induced cells and the non induced cells: there is a shift in fluorescence distribution towards the higher fluorescence for the niacin induced cells. This indicates, as we also observed in the reporter part of the project, that the niacin ligand binds to the receptor. In the third graph we can see that there is a much higher fluorescence value for the cells induced with alpha pheromone. Further we can see that the non induced cells show a fluorescent value, this is what we already had observed in the reporter part of the project: the FUS1 promoter is leaky.

Setup Flow cytometry

In order to test the yeast Snifferomyces NR1 cells with niacin receptor and the receptor inducible GFP reporter we induced the cells with niacin or alpha pheromone and measured the cellular response through flow cytometry, measuring the EGFP signal intensity.


Outcome Flow cytometry

Flow cytometry data of +NR1 cells induced with niacin or alpha pheromones is shown.

Here we see that addition of receptor ligand doesn’t induce an increased light response in comparison with alpha induced cells. Possible explanations are:

  • A too small subset of the yeast receptors is localized in the membrane.
  • The affinity of the receptor with the alpha subunit is too low.

  • Isoamyl acetate Snifferomyces

    Setup

    Since oily undissolved isoamyl acetate can disturb the flow cytomtery experiments a new setup was designed. In this setup we blew air together with gaseous isoamyl acetate trough the yeast culture. In the setup we could measure the offfas CO2 and O2 and we could take samples in time for flow cytometry. The fermenter setup is shown in the figure.

    Outcome

    We runned in total four fermenters: two with the Snifferomyces strain and two with a reference strain. In both cases one of the fermenters was blown with isoamyl acetate and air and the other one only with air. We saw that in both the experiments were the isoamyl acetate was blown trough the fermenters the cells stopped growing. After the isoamyl acetate blow was turned off the cells started to grow again. Thus this experiment tells us that both the reference strain and the Snifferomyces strain cells cannot grow with this high amount of isoamyl acetate.




    Conclusions

    Four olfactory system biobricks were added to the registry. The light response when adding niacin ligand to the NR1 Snifferomyces cells was seen with single cells (fluorescence microscopy), but didn't lead to statistical significant data in the flow cytometry experiment. For the banana receptor Snifferomyces we observed that cells were high concentrations of isoamyl acetate were blown trough, stopped growing.


    Recommendations

    To increase expression and light intensity of the cells in response to a ligand the following conditions can be tested:
    • Make the receptor expression time as short as possible. Use an inducible promoter or make stocks as soon as possible after transformation
    • There are indications that succesful expression can be altered by decreasing the temperature during receptor expression. When we tested this the growth rate was slow and therefore the expression time became too long. An inducible promoter can solve this problem too.
    • The affinity towards the downstream pathway could be increased when the pheromone binding subunit alpha is knocked out (dGPA1) and replaced by a mammalian one (typically NP_034437.1 is used. We didn't have the time to test this hypothesis, further investigation is needed!

    References

    [1] 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)
    [2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443

    Recommendation