Team:TU-Delft/part3

From 2012.igem.org

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<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> 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.<br/>
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> 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.<br/>
<h3>Growth arrest</h3>
<h3>Growth arrest</h3>
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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. Therefor it is needed to knock out the <i>FAR1</i> promoter.<br/>
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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/>
<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]. 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>
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<a name="P9"> <h2>Parts</h2> </a>
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/>
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/>
<img src="http://igem.org/wiki/images/d/d2/GPR109A_snifferomyces.jpg"  width="620" />
<img src="http://igem.org/wiki/images/d/d2/GPR109A_snifferomyces.jpg"  width="620" />
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</p>
</p>
<img src="http://igem.org/wiki/images/6/66/Groeicurve.jpg" align="middle" width="100%">
<img src="http://igem.org/wiki/images/6/66/Groeicurve.jpg" align="middle" width="100%">
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<a name="P9"> <h2>Parts</h2> </a>
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<h3>Transformations</i></h3>
<h3>Transformations</i></h3>

Revision as of 21:31, 26 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]. For this, making a knockout of the native GPA1 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.

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 Δfar1-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 and the R17-ODR10 receptor and output BBa_K775008.

Niacin Snifferomyces

Setup
With yeast strains transformed with the GPR109A receptor and output BBa_K775005 and the R17-ODR10 receptor and output BBa_K775008 a fluorometer experiment was performed. After addition of the ligands (+ control alpha pheromone) OD600 and fluorescence were measured in time.
Outcome

Isoamyl acetate Snifferomyces

Setup
Outcome

Conclusions

Four olfactory system biobricks were added to the registry.


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