Difference between revisions of "Template:BOKU-Vienna/Description"

 
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<h1 id="project-description-">Project Description:</h1>
 
<h1 id="project-description-">Project Description:</h1>
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<h2 id="the-system-is-based-on-a-dcas9-toggle-switch-">The system is based on a dCas9 toggle switch.</h2>
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<p>Our project and the way we see it has evolved constantly since we started working on it. We started in January as we decided that we want to find a better way of protecting crops from pests, disease and harsh environmental conditions. Over time finding a solution against the harsh harvest losses farmers experience every year due to late frost in spring that kills the  blossoms of fruit trees became one of our favourite applications for our system. We wanted to tackle this problem and others with a toggle switch that can control the moment when the trees should bloom. If the genes which control blooming are activated late enough, when late frost is very unlikely to occur, the harvest could be saved. As the project evolved and after a many discussions with people from different fields, it became clear to us, that a gene controlling toggle switch could also be of immense use in numerous fields outside of agriculture, such as plant molecular biology. It could become a universal tool used to toggle any desired gene. It could find applications in foundational research, for example when a scientist wants to study the effects a certain gene has on an organism or in biotechnology to induce the production of a compound later in the fermentation process.
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<p>The ON and OFF gene classes comprise a strong constitutive promoter with an RNA polymerase II binding site and multiple copies of the same gRNA target site downstream next to it.
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There are countless possibilities where a gene-toggling switch would be of use. Since our toggle switch works with a dCas9 system using specific guide RNAs for the activation or deactivation of every single gene, it is possible to have numerous genes that can either be toggled together or independently from each other. This also means, that by activating one toggle switch that produces a specific guide RNA of another toggle switch, the second one is only activated when the first one is activated. Following this idea, it would be possible to create whole logical systems using toggle switches with different guide RNAs. This idea is also the reason why we decided to sign up in the information processing track. We want to create a universal tool which allows us to create a logical switch in cells. Be it simply by starting / shutting down a single gene, or start the expression of a gene only when a complex combination of toggle switches is either activated or deactivated.</p>
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All genes in a class are controlled by the same regulatory region, meaning their gene products are expressed simultaneously.
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At least one gene in each class codes for an RNA construct consisting of a gRNA sequence, flanked by two autonomously folding catalytic RNA sequences, which lead to the production of free gRNA when transcribed.
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Other genes in the ON genes class code for proteins which should be only expressed in the ON state of the system. Vice versa OFF genes encode for OFF state proteins.
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In theory there could be any number and combinations of genes which can define one of the states.
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However, we will only use one additional ON gene and no additional OFF gene.
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The ON gene contains coding sequence for a short lived GFP and will therefore be used as proof of concept.</p>
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<p><br></p>
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<p>Our system has the ability to start or stop the expression of a gene of interest.
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By simply getting activated with the help of a signal molecule once, the state (ON or OFF) will sustain indefinitely, just like a lightbulb.
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This system is based on a so-called toggle-switch which consists of two NOR-gates that use an integrated dCas9 System, where specific guide RNAs bind to a DNA strand and thereby block the transcription.
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<img src="https://static.igem.org/mediawiki/2018/d/db/T--BOKU-Vienna--2018_toggleswitch.png" alt="toggleswitch.png"></p>
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<p><br></p>
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<p>A NOR-gate is the result of the negation of the OR operator, which behaves according to the following truth table.</p>
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<p><br></p>
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<p><table class="table table-dark">
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  <thead>
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        <tr>
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        <th scope="col" colspan="2">Input</th>
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        <th scope="col">Output</th>
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        </tr>
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        <tr>
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        <td>A</td>
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        <td>B</td>
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        <td>A NOR B</td>
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        </tr>
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    </thead>
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  <tbody></tbody></table></p>
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<p><tr></tr></p>
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<p><td>0</td></p>
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<p><td>0</td></p>
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<p><td>1</td>
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</p>
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<p><tr></tr></p>
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<p><td>1</td></p>
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<p><td>0</td></p>
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<p><td>0</td>
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</p>
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<p><tr></tr></p>
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<p><td>0</td></p>
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<p><td>1</td></p>
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<p><td>0</td>
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</p>
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<p><tr></tr></p>
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<p><td>1</td></p>
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<p><td>1</td></p>
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<p><td>0</td>
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<br></p>
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<p>As signals to control the toggle-switch we designed a copper, ethanol and estradiol pathway.</p>
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<p>However, neither of those would work well in agricultural applications.
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Choosing and designing these signal-molecules will be a very important step in future developments of this system, because they should not interfere with the ecosystem.
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As a reporter system for proof of concept in the laboratory we will use a short-lived ubiquitin
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GFP with a half-life of 7 minutes.</p>
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Latest revision as of 23:40, 17 October 2018

Project Description:

Our project and the way we see it has evolved constantly since we started working on it. We started in January as we decided that we want to find a better way of protecting crops from pests, disease and harsh environmental conditions. Over time finding a solution against the harsh harvest losses farmers experience every year due to late frost in spring that kills the blossoms of fruit trees became one of our favourite applications for our system. We wanted to tackle this problem and others with a toggle switch that can control the moment when the trees should bloom. If the genes which control blooming are activated late enough, when late frost is very unlikely to occur, the harvest could be saved. As the project evolved and after a many discussions with people from different fields, it became clear to us, that a gene controlling toggle switch could also be of immense use in numerous fields outside of agriculture, such as plant molecular biology. It could become a universal tool used to toggle any desired gene. It could find applications in foundational research, for example when a scientist wants to study the effects a certain gene has on an organism or in biotechnology to induce the production of a compound later in the fermentation process. There are countless possibilities where a gene-toggling switch would be of use. Since our toggle switch works with a dCas9 system using specific guide RNAs for the activation or deactivation of every single gene, it is possible to have numerous genes that can either be toggled together or independently from each other. This also means, that by activating one toggle switch that produces a specific guide RNA of another toggle switch, the second one is only activated when the first one is activated. Following this idea, it would be possible to create whole logical systems using toggle switches with different guide RNAs. This idea is also the reason why we decided to sign up in the information processing track. We want to create a universal tool which allows us to create a logical switch in cells. Be it simply by starting / shutting down a single gene, or start the expression of a gene only when a complex combination of toggle switches is either activated or deactivated.