Team:TU-Delft/overview

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<h2>Aim</h2>   
<h2>Aim</h2>   
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<p>The goal of this year’s iGEM project is to develop a microbial-based system for the detection of odors, chemicals in gaseous phase. Therefore we will make use of the similarities between the signal transduction cascades of the G-protein coupled receptors (GPCRs) in mammalian cells and the pheromone response pathway in yeast. We aim to functional express mammalian olfactory receptors - GPCRs that bind odorant ligands - in the budding yeast <i>Saccharomyces cerevisiae</i>. By coupling this to a functional reporter it can be used as a novel biosensor for odorant screening.</p><br/>
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<p>The goal of this year’s iGEM project is to develop a microbial-based system for the detection of odors, chemicals in gaseous phase. Therefore we will make use of the similarities between the signal transduction cascades of the G-protein coupled receptors (GPCRs) in mammalian cells and the pheromone response pathway in yeast. We aim to <a href="https://2012.igem.org/Team:TU-Delft/part1#P7">functional express mammalian olfactory receptors</a> - GPCRs that bind odorant ligands - in the budding yeast <i>Saccharomyces cerevisiae</i>. By coupling this to a functional reporter it can be used as a novel biosensor for odorant screening. We characterized three mammalian olfactory receptors: one niacin receptor an two isoamyl acetate (banana smell) receptors. <br>We divided the project in three subparts: <a href="https://2012.igem.org/Team:TU-Delft/part1">Receptor</a>,<a href="https://2012.igem.org/Team:TU-Delft/part2"> Reporter</a> and <a href="https://2012.igem.org/Team:TU-Delft/part3">Snifferomyces</a> (=receptor+reporter).</p>
<h2>yeast</h2>   
<h2>yeast</h2>   
<h3>Yeast, we choose you!</h3>
<h3>Yeast, we choose you!</h3>
<p>For this project, we will use <i>S. cerevisiae</i> as a host organism because it utilizes already a GPCR pathway: the mating pathway. </p>
<p>For this project, we will use <i>S. cerevisiae</i> as a host organism because it utilizes already a GPCR pathway: the mating pathway. </p>
<h3>Sex response of <i>S. cerevisiae</i></h3>
<h3>Sex response of <i>S. cerevisiae</i></h3>
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<p>Yeast genders are called 'a' and 'α', and both genders extract pheromones called 'a'- and 'α'-pheromones. The 'a'-yeasts are able to detect the 'α'-pheromones, and so the other way around. Upon detection, the yeast cells will will go in growth arrest and prepare for mating. This mating response can be seen in the form of a morphological change, called shmoo formation.</p>
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<p>Yeast genders are called 'a' and 'α', and both genders extract pheromones called 'a'- and 'α'-pheromones. The 'a'-yeasts are able to detect the 'α'-pheromones, and so the other way around. Once the pheromone receptors detects pheromones of another gender, the G-alpha subunit comes to action, dissociating from the GPCR complex. This protein starts a signal leading to growth arrest and to a mating response, of which the morphology is called a shmoo.<br>
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<p style="color:#2ab118;">The image below links to the page explaining more about yeast and why we decided to use it!</p>
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<a href="https://2012.igem.org/Team:TU-Delft/Yeast" rel="lightbox" title="shmoo">
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<img src="https://static.igem.org/mediawiki/igem.org/b/b3/Shmooooo.jpg" name="kugroup" width="400"  border="0" id="kugroup" /></a></div>
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<h2>Subparts</h2>
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<h3>Receptor</h3><h6>I'd appreciate your input!</h6>
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<p>Once the pheromone-receptors detects pheromones of another gender, the G-alpha subunit comes to action, dissociating from the GPCR complex. This protein starts a signal leading to growth arrest and to a mating response, of which the morphology is called a shmoo. To make the smelling device to detect tuberculosis, our yeast cells need a Methyl nicotinate- (the scent associated with tuberculosis) receptor. Unfortunately a methyl nicotinate receptor isn't characterized as such yet and we chose a very related ligand receptor: niacin. This receptor, due to it's <a href="https://2012.igem.org/Team:TU-Delft/part1#A1">chimeric properties</a> is transported to the same place as the pheromone receptor and will use the same pathway. In the end our goal is to see whether we can change this receptor through <a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling">directed mutagenesis</a> to let it smell methyl nicotinate.</p>
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<p style="color:#2ab118;"> To view the plasmids we have made and the accompanying experiments you can click the image below.</p>  
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<a href="https://2012.igem.org/Team:TU-Delft/part1" rel="lightbox" title="shmoo">
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<img src="https://static.igem.org/mediawiki/igem.org/b/b3/Shmooooo.jpg" width="53%" height="35%"
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<img src="https://static.igem.org/mediawiki/igem.org/3/31/Receptors_general.jpg" name="kugroup" width="500" border="0" id="kugroup" /></a></div>
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<h2>Subparts</h2>
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<h3>Receptor</h3>
<h3>Receptor</h3>
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<p>When the cell detects a smell, niacin in this case, we do not want the cell growth to stop, so we deleted the far1-gene, which causes growth arrest.  
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<p>To make the smelling device to detect the ligands that we choose (tubercolosis smell and bananasmell) our yeast cells need to express the corresponding receptors. The receptors, due the <a href="https://2012.igem.org/Team:TU-Delft/part1#A1">chimeric properties</a> that we gave them, are transported to the same place as the pheromone receptor and will use the same pathway.</p>
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Upon detecting this niacin molecule, we would like to see more than a mating response, the shmoo. For this reason, we added a <a href="https://2012.igem.org/Team:TU-Delft/part2#A2">GFP-output</a> which is promoted by the mating response inducible promoter, <a href="https://2012.igem.org/Team:TU-Delft/part2#A3"> FUS1</a>. </p>  
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<img src="https://static.igem.org/mediawiki/igem.org/8/88/Receptorchillplaatje.png" width="40%" height="35%"
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<h3>Reporter</h3>
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<p>Upon detecting the ligand molecule, we would like to see more than a shmoo mating response. For this reason, we added a <a href="https://2012.igem.org/Team:TU-Delft/part2#A2">EGFP-output</a> which is promoted by the mating response inducible promoter, <a href="https://2012.igem.org/Team:TU-Delft/part2#A3"> <i>FUS1</i></a>.</p>
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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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<h3>Snifferomyces</h3>
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<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained: the snifferomyces. If the corresponding ligand binds to the receptor the underlying cascade is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. <br/>
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When the cell detects a ligand we do not want the cell to stop growing, so we deleted the <i>FAR1</i> gene, which causes growth arrest.</p>
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<img src="https://static.igem.org/mediawiki/igem.org/e/ee/Outputeninput.png" width="40%" height="35%"
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<p style="color:#2ab118;">Click the image to learn more about our GFP-output!</p>
 
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<a href="https://2012.igem.org/Team:TU-Delft/part2" rel="lightbox" title="shmoo">
 
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<h3>Olfactory system</h3>
 
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<p>Our product is a yeast cell which can detect methyl nicotinate, associated with tuberculosis, and shows a <a href="https://2012.igem.org/Team:TU-Delft/part2#A2">GFP-output</a> upon this detection. We have also experimented with the banana smell methyl nicotinate and a couple of other smells with which the Hong Kong team has experimented before us.</p>
 
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Latest revision as of 10:27, 20 December 2012

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Receptor

Aim

The goal of this year’s iGEM project is to develop a microbial-based system for the detection of odors, chemicals in gaseous phase. Therefore we will make use of the similarities between the signal transduction cascades of the G-protein coupled receptors (GPCRs) in mammalian cells and the pheromone response pathway in yeast. We aim to functional express mammalian olfactory receptors - GPCRs that bind odorant ligands - in the budding yeast Saccharomyces cerevisiae. By coupling this to a functional reporter it can be used as a novel biosensor for odorant screening. We characterized three mammalian olfactory receptors: one niacin receptor an two isoamyl acetate (banana smell) receptors.
We divided the project in three subparts: Receptor, Reporter and Snifferomyces (=receptor+reporter).

yeast

Yeast, we choose you!

For this project, we will use S. cerevisiae as a host organism because it utilizes already a GPCR pathway: the mating pathway.

Sex response of S. cerevisiae

Yeast genders are called 'a' and 'α', and both genders extract pheromones called 'a'- and 'α'-pheromones. The 'a'-yeasts are able to detect the 'α'-pheromones, and so the other way around. Once the pheromone receptors detects pheromones of another gender, the G-alpha subunit comes to action, dissociating from the GPCR complex. This protein starts a signal leading to growth arrest and to a mating response, of which the morphology is called a shmoo.

Subparts

Receptor

To make the smelling device to detect the ligands that we choose (tubercolosis smell and bananasmell) our yeast cells need to express the corresponding receptors. The receptors, due the chimeric properties that we gave them, are transported to the same place as the pheromone receptor and will use the same pathway.

Reporter

Upon detecting the ligand molecule, we would like to see more than a shmoo mating response. For this reason, we added a EGFP-output which is promoted by the mating response inducible promoter, FUS1.

Snifferomyces

By combining the olfactory receptor and the FUS1pr-EGFP reporter, a complete yeast olfactory system is obtained: the snifferomyces. If the corresponding ligand binds to the receptor the underlying cascade is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter.
When the cell detects a ligand we do not want the cell to stop growing, so we deleted the FAR1 gene, which causes growth arrest.