Latest revision as of 03:35, 29 October 2013

Reporter

In order to show when our detector TALEs are bound to DNA, we had to find a suitable reporter component. After careful consideration, we set upon an enzyme/catalyst reporter because preliminary modelling with fluorescent proteins would not provide a strong, sustained output for our intended prototype. Fluorescent proteins would also require a complex prototype to excite the protein with specific wavelengths of light. In order to decrease complexity and cost of our device, we needed to a reporter with a higher output per reporter unit.

Based on conversations with industry and manufacturing criteria, we realized that our reporter would have to meet three pieces of criteria. Firstly, it would require rapid enzyme kinetics so that it could keep pace with production in meat processing plants and feedlots. Secondly, we needed a reporter with a visible colour output so that we could integrate it into a lateral flow prototype interpretable without unwieldy laboratory equipment. Finally, we needed an enzyme which could be manufactured in E. coli that would be durable in the field and cost effective.

Through some of our initial modelling, we compared several enzymes and found horse radish peroxidase. We realized, however, that horse radish peroxidase could not be expressed in a prokaryotic system. To solve this problem, we decided to use Prussian blue chemically modified ferritin, which could induce similar colour changes in horse radish peroxidase substrates. Additionally, we were intrigued by how Prussian blue ferritin could be used to scaffold multiple detector TALEs to modulate sensitivity of the overall system. Through our literature searches, we also identified β-lactamase as a monomeric reporter which could be readily expressed in E. coli. Thus, we set out to characterize two reporter systems, both of which were show to have their own advantages, to enable us to adapt our system for the beef industry. Learn more about these reporters below:

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 <h1>Reporter</h1>
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-<p>For this project it was necessary to select a method by which our system could inform the user of a positive test result, that is, we needed a reporter system. After careful consideration, we decided upon an <span class="Green"><b>enzyme/catalyst reporter</b></span> as we believed that using a reporter such as green fluorescent protein (GFP) simply would not provide a strong enough of a signal for a portable device. GFP would provide a constant signal that would not increase in intensity over time. The fluorescent output of GFP would also require the device to have the ability to excite the protein with the appropriate wavelength of light. This would add to both the complexity and cost of the device, making it harder to offer a portable and cheap solution.</p>
+<p>In order to show when our detector TALEs are bound to DNA, we had to find a suitable reporter component. After careful consideration, we set upon an <span class="Yellow"><b>enzyme/catalyst reporter</b></span> because preliminary modelling with fluorescent proteins would not provide a strong, sustained output for our intended prototype. Fluorescent proteins would also require a complex prototype to excite the protein with specific wavelengths of light. In order to decrease complexity and cost of our device, we needed to a reporter with a higher output per reporter unit.</p>
-<p>We decided that an enzyme with a colour output would be more suitable and easier to implement in the field. Many common enzymes were analyzed for this purpose. One example is <span class="Green"><b>horseradish peroxidase.</span></b> This enzyme has high enzymatic activity and is commonly used in many biological applications. One problem with its use however is that it cannot be effectively produced within <i>E. coli</i>. There are other common enzymes that do not have this issue such as alkaline phosphatase. These enzymes however are often multimeric which presents an issue with our in vitro strip system. If the enzymes are not able to assemble correctly with the rest of our proteins, the device will be ineffective. Upon further investigation, we found two candidates: the ampicillin resistance enzyme <span class="Green"><b>beta-lactamase</span></b> and <span class="Green"><b>ferritin</span></b>, a protein which can be chemically modified to become a strong catalyst. Why two different reporters? Characterizing two reporter systems, both of which have their own key advantages, gives us <span class="Green"><b>flexibility</span></b> in our platform technology to meet the different criteria that <i> will </i> exist for different detection systems. In addition, during our characterization, if one reporter does not act as it is meant to, we have the ability to focus on the other reporter for our system. Click the links below to learn more about these two systems:</p>
+<p>Based on conversations with industry and manufacturing criteria, we realized that our reporter would have to meet three pieces of criteria. Firstly, it would require rapid enzyme kinetics so that it could keep pace with production in <a href="https://2013.igem.org/Team:Calgary/Project/HumanPractices/InformedDesign">meat processing plants and feedlots</a>. Secondly, we needed a reporter with a visible colour output so that we could integrate it into a <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Prototype">lateral flow prototype</a> interpretable without unwieldy laboratory equipment. Finally, we needed an enzyme which could be manufactured in <i>E. coli</i> that would be durable in the field and cost effective.</p>
+<p>Through some of our <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Modelling/QuantitativeModelling">initial modelling</a>, we compared several enzymes and found horse radish peroxidase.  We realized, however, that horse radish peroxidase could not be expressed in a prokaryotic system. To solve this problem, we decided to use <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">Prussian blue chemically modified ferritin</a>, which could induce similar colour changes in horse radish peroxidase substrates. Additionally, we were intrigued by how Prussian blue ferritin could be used to scaffold multiple detector TALEs to modulate sensitivity of the overall system. Through our literature searches, we also identified <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">&#946;-lactamase</a> as a monomeric reporter which could be readily expressed in <i>E. coli</i>.  Thus, we set out to characterize two reporter systems, both of which were show to have their own advantages, to enable us to adapt our system for the beef industry. Learn more about these reporters below:</p>
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 <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">
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 <h2>Prussian-Blue Ferritin</h2>
-<img src="https://static.igem.org/mediawiki/2013/9/93/2013UCalgaryButtonFerritin.png">
+<img src="https://static.igem.org/mediawiki/2013/5/59/2013calgarybuttonsnewPrussianblueferritin.png">
-<p>Combining the power of chemistry and biology we have successfully converted the iron-storage nanoparticle Ferritin into a robust and stable peroxidase-like catalyst. Click here to learn more!</p>
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 <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">
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-<h2>Beta-Lactamase</h2>
+<h2>&#946;-Lactamase</h2>
-<img src="https://static.igem.org/mediawiki/2013/f/f2/2013UCalgaryButtonBetaLac.png">
+<img src="https://static.igem.org/mediawiki/2013/0/06/2013calgarybuttonsforwikinewBetalactamase.png">
-<p>A small, monomeric enzyme that normally confers antibiotic resistance as a reporter enzyme? Click here to delve more into our research about the uses of this enzyme as a reporter in our system!</p>
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