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Team:Calgary/Project/OurSensor/Reporter
From 2013.igem.org
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<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 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 | + | <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 suggested that such proteins would not provide a strong, sustained output for our intended prototype. Fluorescent proteins would provide a constant signal which would an increasing output over time. 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 unit of reporter.</p> |
<p>Based on conversations with industry and manufacturing criteria, we realized that our reporter would have to meet three pieces of criteria. Firstly, our reporter would have to have rapid enzyme kinetics so that it could keep pace with production in meat processing plants and feedlot. Secondly, we needed a reporter with a visible colour output so that we could integrate it into a lateral flow prototype which could be interpreted 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.</p> | <p>Based on conversations with industry and manufacturing criteria, we realized that our reporter would have to meet three pieces of criteria. Firstly, our reporter would have to have rapid enzyme kinetics so that it could keep pace with production in meat processing plants and feedlot. Secondly, we needed a reporter with a visible colour output so that we could integrate it into a lateral flow prototype which could be interpreted 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.</p> | ||
Revision as of 02:50, 29 October 2013
Reporter
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 suggested that such proteins would not provide a strong, sustained output for our intended prototype. Fluorescent proteins would provide a constant signal which would an increasing output over time. 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 unit of reporter.
Based on conversations with industry and manufacturing criteria, we realized that our reporter would have to meet three pieces of criteria. Firstly, our reporter would have to have rapid enzyme kinetics so that it could keep pace with production in meat processing plants and feedlot. Secondly, we needed a reporter with a visible colour output so that we could integrate it into a lateral flow prototype which could be interpreted 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 were also identified beta-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:
