Difference between revisions of "Team:Berlin diagnostX/Design"

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    <h1 class="text-center head_us head-light">Design</h1>
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<div class="abstand_navbar headimage" id="background_description_head"> <h1 class="text-center head_us head-light">Design</h1> </div>
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             <h1 class="text-center igem_blue">The vision of diagnost-x</h1>
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            <h4 class="text-center">Developing a low cost, easy-to-handle diagnostic test for infection with the tapeworm Taenia solium</h4>
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             <h3 class="text-center igem_blue mt-4"><strong>Establishing the screening pipeline</strong></h3>
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             <h5 class="text-left igem_blue pb-1">Optimizing the Cell Free Expression System </h5>
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             <h5 class="igem_blue text-center pb-1">Transcriptome Analysis of T. solium</h5>
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             <p class="text-justify">The <em>PureExpress®-System </em> is composition of completely recombinant molecules to effectively transcribe any form of DNA and translate the produced mRNA.
 <br> To use this system for field diagnostics we figured that it has to meet certain requirements:</p>
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             <p class="text-center">Behind the Bioinformatics</p>
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<ul>
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<li> Heat stability up to a temperature of 50° C</li>
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<li> High efficiency in low volumes and smaller concentrations</li>
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<li> Stability towards change in electrolyte concentration and addition of foreign molecules</li>
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<li> Storability</li>
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</ul>
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<p class="text-justify">We successfully verified the outstanding function of the system under these conditions.
Especially noteworthy, we were able to create significant color change in a volume of 2,5 uL which is 10% of the volume recommended by the manufacturer. Furthermore, we could show that the system is still highly active in concentrations of 40-50%. This makes it possible to elute it with patient samples without tempering with its functionality. 
Last but not least, the system is not very vulnerable to a fair amount of possibly interfering molecules like for example the chlorphenol-red we use as reporter assay.
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We concluded that the PureExpress® - System is most suitable for our essay; also due to the fact, that it composed of recombinant parts instead of cell lysate and so has very few variability.
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             <div class="text-center"><a class="btn btn-lg igem_but btn-primary red" href="#bioinfo" role="button">Discover More</a></div>
 
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             <h5 class="text-left igem_blue">RNA sequencing </h5>
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             <div class="text-center"><a class="btn btn-lg igem_but btn-primary red" href="#teamorga" role="button">Discover More</a></div>
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            <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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             <h3 class="text-center igem_blue mt-4"><strong>The bioinformatics behind our switch design</strong></h3>
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             <h5 class="text-center pb-1"><strong>Transcriptome Analysis of T. solium</strong></h5>
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            <p class="text-justify">Irgendn Text</p>
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            <h5 class="text-center pb-1"><strong>Noch ne Überschrift</strong></h5>
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            <p class="text-justify">Irgendn Text</p>      
 
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            <p class="lead text-center">In order to establish our diagnostic test we thus needed:</p>
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            <ul>
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                <li>An <strong>RNA sensor</strong> designed to target the tapeworm</li>
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                <li>A <strong>cell free expression system</strong> able to produce a color change</li>
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                <li>T. solium <strong>specific trigger RNA</strong></li>
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                <li>(for field use: a carrier system to make the cell-free expression system storable)</li>
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            </ul>
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            <p class="lead text-center mt-3" >We thus divided the work over three subteams:</p>
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            <ol>
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                <li><a class="igem_blue" href="#team0">In-silico RNA sensor design (Team 0)</a></li>
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                <li><a class="igem_blue" href="#team1">Sensor synthesis (Team 1) </a></li>
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                <li><a class="igem_blue" href="#team2">Sensor Screening/Cell free expression (Team 2)</a></li>
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            </ol>
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        <div class="col-12"><h3 class="text-center igem_blue mb-3">Preliminary Data (Bioinformatics)</h3></div>
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             <h5 class="text-left igem_blue pb-1">In silico design </h5>
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            <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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             <p class="text-justify">Blablabla</p>
 
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         <div class="col-12"><h3 class="text-center igem_blue mb-3">Team 0: In-silico RNA sensor design</h3></div>
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            <h3 class="text-center igem_blue mt-4"><strong>The switch synthesis</strong></h3>
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             <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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             <p class="text-center">Text dazu</p>
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             <h5 class="text-left igem_blue">Extension PCR </h5>
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             <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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        <div class="col-6"><img src="https://static.igem.org/mediawiki/2017/a/a8/T--Berlin_diagnostX--Switch_detailed.png" class="img-fluid"></div>
 
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             <h5 class="text-left igem_blue pb-1">Nested PCR</h5>
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             <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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             <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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             <p class="text-center">Text dazu</p>
 
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             <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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            <p class="text-center">Text dazu</p>
 
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        <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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             <h5 class="text-left igem_blue">Colony PCR </h5>
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             <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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             <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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             <p class="text-center">Text dazu</p>
 
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            <h5 class="text-left igem_blue pb-1">DNA Clean Up (via column & gel extraction) </h5>
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            <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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        <div class="col-12"><h3 class="text-center igem_blue mb-3">Team 1: Sensor Synthesis </h3></div>
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             <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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            <p class="text-center">Text dazu</p>
 
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        <div class="col-6"><img src="https://static.igem.org/mediawiki/2017/b/b7/T--Berlin_diagnostX--Extension_PCR.JPG" class="img-fluid"></div>
 
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             <h5 class="text-left igem_blue">Gel electrophoresis </h5>
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             <h5 class="igem_blue text-center pb-1">Zwischenüberschrift</h5>
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             <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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         <div class="col-12"><h3 class="text-center igem_blue mb-3">Team 2: Sensor Screening/Cell free expression system</h3></div>
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            <h3 class="text-center igem_blue mt-4"><strong>The Idea</strong></h3>
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            <h5 class="text-left igem_blue pb-1">The Tapeworm <em>Taenia solium</em> </h5>
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             <p class="text-center">Text dazu</p>
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             <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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            <p class="text-justify">The test will be based on a newly developed method called toehold switch sensors. This method has already been successfully applied to the diagnosis of the Zika virus [3]. We are therefore convinced that toehold switch sensors will also be useful in detecting T. solium.
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            In order to develop our test, we first need to achieve two things:
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            The test will be based on a newly developed method called toehold switch sensors. This method has already been successfully applied to the diagnosis of the Zika virus [3]. We are therefore convinced that toehold switch sensors will also be useful in detecting T. solium.
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            In order to develop our test, we first need to achieve two things:
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            <p class="summarybox p-4"><strong>Summary</strong>: Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum.</p>
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            <h3 class="text-center mb-1"><strong>Summary</strong></h3>
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            <p class="text-justify">In order to develop a diagnostic tool for the tapeworm infection Taeniasis, we developed a pipeline that allowed for designing, synthesizing and testing any given sensor within 2 days. <br> <strong>Step 1: In silico design</strong></p>
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            <ul>
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                <li>Design of a toehold switch sensor specific to T. solium RNA</li>
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                <li>Generating primers necessary for switch synthesis</li>
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            </ul>
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            <p class="text-justify"><strong>Step 2: DNA-Switch Synthesis</strong></p>
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            <ul>
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                <li>By using extension PCR or nested PCR, the toehold switches were synthesized</li>
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                <li>Reporter gene (Beta-galactosidase) and T7 promoter were added to the sensor</li>
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            </ul>
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            <p class="text-justify"><strong>Step 3: Sensor Screening</strong></p>
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            <ul>
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                <li>Transcription of DNA sensor into RNA takes place in CFE system</li>
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                <li>Specific binding of RNA sensor to trigger RNA leads to translation of reporter gene in CFE system</li>
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                <li>By measuring absorption at 560 nm, color change was measured and enzymatic reaction quantified</li>
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            <p class="text-justify">Splitting the work into three teams enabled us to work continuously – trouble shooting in one team did not stall work in the other teams. Alongside the work on the core piece – a CFE system containing a sensitive and specific sensor – we tried to keep the ultimate goal in mind: a test usable in the field. <br><br> We thus investigated fixing the system onto cellulose membranes, making it storable, durable and easily transportable. Collaborations with partners in Berlin, Europe and India enabled us to further adapt our system to challenges of real-life application.</p>
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            <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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            <h5 class="text-left igem_blue pb-1">Toehold Switch Sensor Test</h5>
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            <p class="text-justify"><em>Taenia solium</em>, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point <a class="igem_blue" href="#"> [1]</a>. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis. </p>
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                 <img src="https://static.igem.org/mediawiki/2017/4/4f/T--Berlin_diagnostX--toehold_easy.png" class="img-fluid">
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                 <h5 class="text-center">References</h5>
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[1] Pardee, K. et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 165, 1255–1266 (2016). <br>
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[2] Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell 159, 925–939 (2014) <br>
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[3] Gomez, S. et al. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR). Sci. Rep. 5, 9683 (2015)
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            <p class="text-justify">The synthetic biology experiments described here are particularly important for participation in the iGEM competition. Simultaneously, a second team will work on the recovery of T. solium-RNA from patient stool samples in order to make our test applicable for medical purposes. We are currently forming the necessary partnerships for this goal: A working group in Nairobi has already committed to providing us with relevant material. However, these experiments will be conducted in a properly equipped laboratory, independent from the previously described experiments.</p>
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{{Berlin_diagnostX2}}
 

Revision as of 18:18, 1 November 2017

Design

The vision of diagnost-x

Developing a low cost, easy-to-handle diagnostic test for infection with the tapeworm Taenia solium

Transcriptome Analysis of T. solium

Behind the Bioinformatics

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Transcriptome Analysis of T. solium

Irgendn Text

Noch ne Überschrift

Irgendn Text

In order to establish our diagnostic test we thus needed:

  • An RNA sensor designed to target the tapeworm
  • A cell free expression system able to produce a color change
  • T. solium specific trigger RNA
  • (for field use: a carrier system to make the cell-free expression system storable)

We thus divided the work over three subteams:

  1. In-silico RNA sensor design (Team 0)
  2. Sensor synthesis (Team 1)
  3. Sensor Screening/Cell free expression (Team 2)

Preliminary Data (Bioinformatics)

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Team 0: In-silico RNA sensor design

Zwischenüberschrift

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Team 1: Sensor Synthesis

Zwischenüberschrift

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Team 2: Sensor Screening/Cell free expression system

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Summary

In order to develop a diagnostic tool for the tapeworm infection Taeniasis, we developed a pipeline that allowed for designing, synthesizing and testing any given sensor within 2 days.
Step 1: In silico design

  • Design of a toehold switch sensor specific to T. solium RNA
  • Generating primers necessary for switch synthesis

Step 2: DNA-Switch Synthesis

  • By using extension PCR or nested PCR, the toehold switches were synthesized
  • Reporter gene (Beta-galactosidase) and T7 promoter were added to the sensor

Step 3: Sensor Screening

  • Transcription of DNA sensor into RNA takes place in CFE system
  • Specific binding of RNA sensor to trigger RNA leads to translation of reporter gene in CFE system
  • By measuring absorption at 560 nm, color change was measured and enzymatic reaction quantified

Splitting the work into three teams enabled us to work continuously – trouble shooting in one team did not stall work in the other teams. Alongside the work on the core piece – a CFE system containing a sensitive and specific sensor – we tried to keep the ultimate goal in mind: a test usable in the field.

We thus investigated fixing the system onto cellulose membranes, making it storable, durable and easily transportable. Collaborations with partners in Berlin, Europe and India enabled us to further adapt our system to challenges of real-life application.

References

[1] Pardee, K. et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 165, 1255–1266 (2016).
[2] Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell 159, 925–939 (2014)
[3] Gomez, S. et al. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR). Sci. Rep. 5, 9683 (2015)