Team:Calgary/Project/OurSensor

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<h1>Our Sensor</h1>
<h1>Our Sensor</h1>
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<p> class="noIndent">The goal of our project is to design a biosensor to rapidly identify cattle known as <span class ="Green"><b>super shedders.</b></span>  Super shedders are cattle that excrete extremely large numbers of <i>E.coli</i> O157:H7 a subgroup of <i>E. coli</i> referred to as Enterohemorrhagic <i>E. coli</i> (EHEC). EHEC organisms produce a toxin called <span class="Green"><b>Shiga toxin</span></b> or verotoxin. This toxin binds to blood cells and lyses them resulting in hemolytic-uremic syndrome (HUS) in humans and can be <span class="Green"><b>deadly. </b> Supershedder excrete <i> E. coli </i> O157:H7 in the range of 10 <sup>7</sup> to 10 <sup>10 </sup> exceeding normal cattle by 3-6 logs and can contaminate other cattles in the same holding penn as well as the meat downstream. Supershedders are thought to be the reason for 95% of the <i>E. coli</i> O157:H7 contamination in the industry.</span></b></p>
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<p>The goal of our project is to design a biosensor to rapidly identify cattle known as <span class="Yellow"><b>super shedders.</b></span>  Super shedders are cattle that excrete extremely large numbers of <i>E.coli</i> O157:H7 a subgroup of <i>E. coli</i> referred to as Enterohemorrhagic <i>E. coli</i> (EHEC). EHEC organisms produce a toxin called <span class="Yellow"><b>Shiga toxin</span></b> or verotoxin. This toxin binds to renal cells and lyses them, resulting in hemolytic-uremic syndrome (HUS) in humans and can be <span class="Yellow"><b>deadly</span></b>. Supershedders excrete more than 10<sup>4</sup> colony forming units of <i> E. coli </i> O157:H7 in every gram of their feces and can contaminate other cattle in the same holding pen as well as the meat downstream. Super-shedders are thought to be the reason for 95% of the <i>E. coli</i> O157:H7 contamination in the industry.</b></p>
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<p>We are building a <span class="Green"><b>DNA-based biosensor</span></b> that specifically detects the gene called <i>stx2</i> that is common to all EHEC organisms.  Our device will be useful for the detection of not only the O157:H7 strain of <i>E.coli</i> but for the other EHEC strains as well. Click on the components below to learn more about their design and function. View our <span class="Green"><b>animation</span></b> below to see how the system would actually work!</p>
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<p>We are building a <span class="Yellow"><b>DNA-based biosensor</span></b>, as it is more reliable and cheaper than a protein-based sensor; antibodies are expensive and the proteins that they target can get degraded during the sample preparation, whereas DNA is much more stable. A DNA-based sensor also enabled us to target a broader range of harmful <i>E.coli</i>. Our sensor is specific to shiga toxin (stx2) gene, present not only in <i>E.coli</i> O157:H7, but other EHEC strains as well. This means our detector will not only be specific to one specific strain of EHEC, but instead target a broad group of EHEC organisms. Click on the components below to learn more about their design and function. View our <a href="#animation">animation</a> below to see how the system would actually work!</p>
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<p>To see how our system would work as a platform in industry and in the registry, please click <a href="https://2013.igem.org/Team:Calgary/Project/HumanPractices/Platform">here</a>.
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<h2>The FerriTALE System:</h2>
<h2>The FerriTALE System:</h2>
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<p> As seen in the <a href="https://static.igem.org/mediawiki/2013/e/e3/UCalgary-2013-Ferritale-Closeup.mp4" target="_blank"><span class="green"><b>video</b></span></a> above our goal is to develop a strip based assay that can alert us upon detection of pathogenic DNA sequence, namely <i>Stx 2</i>. We will immobilize a TALE-Ferritin scaffold (<span class="Green"><b>a FerriTALE!</span></b>) to a nitrocellulose strip. The TALE protein that has been immoblized will bind to a unique oligonucleotide sequence that is approximately 18-20 base pairs in size on the <i>Stx 2</i> gene. This <span class="Green"><b>immobile component</span></b> of our system (Figure 1) will effectively capture our target DNA on the strip. A second TALE attached to our reporter (Prussian blue ferritin or &beta;-lactamase) that targets a different unique sequence on the <i> Stx 2</i> gene will then be run along the nitrocellulose as a <span class="Green"><b>mobile component</span></b>. The TALE-reporter complex will only bind if the second target DNA sequence is present. Following this we will add a substrate solution and if both of the TALE proteins are bound to the <i>Stx 2</i> gene we will see a <span class="Green"><b>colour output</span></b> on the strip.</p>
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<p> As seen in the <a href="https://static.igem.org/mediawiki/2013/e/e3/UCalgary-2013-Ferritale-Closeup.mp4" target="_blank">video</a> above our goal is to develop a strip based assay that can alert us upon detection of pathogen specific DNA sequence, namely <i>stx2</i>. We will treat our sample collected from the supershedders with a TALE-Ferritin complex  (<span class="Yellow"><b>a FerriTALE!</span></b>). This DNA-TALE-Ferritin complex would then be flown over our strip that contains a second, immobilized TALE that recognizes yet another 18-20 bp region on the <i>stx 2</i> gene. This second TALE will immobilize our DNA-TALE-ferritin complex on the strip. Following immobilization we will add substrate to our strip. Only in the presence of both the EHEC sequences we get a colour change on the strip. If there is only one of the sequences present, the colour change does not occur either due to lack of immobilization or due to lack of presence of a reporter.</p>
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<p><b>Figure 1.</b> Our system is composed of both an immobile element on the strip that will capture our target DNA and a mobile element that will report the presence of our target DNA. </p>
<p><b>Figure 1.</b> Our system is composed of both an immobile element on the strip that will capture our target DNA and a mobile element that will report the presence of our target DNA. </p>
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<p>TALEs are modular and can be used <span class="Green"><b>platform technology</span></b> implying TALEs can be engineered to detect any DNA of interest. We are using TALE proteins as our sensory element. TALEs are very powerful tools since they can be engineered to bind to any 12 to 31bp sequence of DNA (source: Golden Gate TALEN and TAL Effector Kit 2.0). Therefore, by just swapping the TALEs in our PLASMID XYZ, one can detect any DNA of interest. To further ease this process, we have incorporated a KasI restriction cut site at the end of all TALEs in all the constructs. Using the KasI restriction enzyme and one of the biobrick prefix enzymes one can incorporate their own engineered TALE into the part, and use it for detecting their DNA of interest.</p>
 
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Latest revision as of 03:27, 29 October 2013

Our Sensor

The goal of our project is to design a biosensor to rapidly identify cattle known as super shedders. Super shedders are cattle that excrete extremely large numbers of E.coli O157:H7 a subgroup of E. coli referred to as Enterohemorrhagic E. coli (EHEC). EHEC organisms produce a toxin called Shiga toxin or verotoxin. This toxin binds to renal cells and lyses them, resulting in hemolytic-uremic syndrome (HUS) in humans and can be deadly. Supershedders excrete more than 104 colony forming units of E. coli O157:H7 in every gram of their feces and can contaminate other cattle in the same holding pen as well as the meat downstream. Super-shedders are thought to be the reason for 95% of the E. coli O157:H7 contamination in the industry.

We are building a DNA-based biosensor, as it is more reliable and cheaper than a protein-based sensor; antibodies are expensive and the proteins that they target can get degraded during the sample preparation, whereas DNA is much more stable. A DNA-based sensor also enabled us to target a broader range of harmful E.coli. Our sensor is specific to shiga toxin (stx2) gene, present not only in E.coli O157:H7, but other EHEC strains as well. This means our detector will not only be specific to one specific strain of EHEC, but instead target a broad group of EHEC organisms. Click on the components below to learn more about their design and function. View our animation below to see how the system would actually work!

To see how our system would work as a platform in industry and in the registry, please click here.

The FerriTALE System:



As seen in the video above our goal is to develop a strip based assay that can alert us upon detection of pathogen specific DNA sequence, namely stx2. We will treat our sample collected from the supershedders with a TALE-Ferritin complex (a FerriTALE!). This DNA-TALE-Ferritin complex would then be flown over our strip that contains a second, immobilized TALE that recognizes yet another 18-20 bp region on the stx 2 gene. This second TALE will immobilize our DNA-TALE-ferritin complex on the strip. Following immobilization we will add substrate to our strip. Only in the presence of both the EHEC sequences we get a colour change on the strip. If there is only one of the sequences present, the colour change does not occur either due to lack of immobilization or due to lack of presence of a reporter.

System Elements

Figure 1. Our system is composed of both an immobile element on the strip that will capture our target DNA and a mobile element that will report the presence of our target DNA.