Team:Calgary/Project/OurSensor/Reporter

From 2013.igem.org

(Difference between revisions)
 
(11 intermediate revisions not shown)
Line 8: Line 8:
<p><img style="float: right;" src="https://static.igem.org/mediawiki/2013/8/81/2013ccalgaryfigurefortheReporterpage.png" width="25%" height="25%"></p>
<p><img style="float: right;" src="https://static.igem.org/mediawiki/2013/8/81/2013ccalgaryfigurefortheReporterpage.png" width="25%" height="25%"></p>
-
<p>In order to show when our detector TALEs have are bound to target DNA, we had to find an suitable reporter component. After careful consideration, we set upon an <span class="Yellow"><b>enzyme/catalyst reporter</b></span> as our preliminary modelling of green fluorescent protein (GFP) output indicated it would not provide a strong enough 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. In order to decrease complexity and cost of our device, we needed to a reporter with a higher output per unit of reporter.</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 deploy in a field setting. A coloured output could be integrated into a simple, visual output. From our conversations with a feedlot, we realized we'd require fast reporter kinetics so that our system could keep pace with industry production. Additionally, we also In our preliminary modelling, we tested common reporters including beta-galactosidase. One example is <span class="Yellow"><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 <i>in vitro</i> strip system.  If the enzymes are not able to assemble correctly with the rest of our proteins, the device will be ineffective. We also modelled the activities of some of these common reporters based on literature review in order to aid in informing us of the appropriate reporter for our system. Upon further investigation, we found <span class="Yellow"><b>two candidates</span></b></class>: the ampicillin resistance enzyme <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">beta-lactamase</a> and <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin">ferritin</a>, 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="Yellow"><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>
<section id="ButtonsSection">
<section id="ButtonsSection">
Line 23: Line 25:
<a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">
<a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">
<div id="Button2" class="Button" style="height: 388px;">
<div id="Button2" class="Button" style="height: 388px;">
-
<h2>Beta-Lactamase</h2>
+
<h2>&#946;-Lactamase</h2>
<img src="https://static.igem.org/mediawiki/2013/0/06/2013calgarybuttonsforwikinewBetalactamase.png">
<img src="https://static.igem.org/mediawiki/2013/0/06/2013calgarybuttonsforwikinewBetalactamase.png">
</div>
</div>

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: