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Team:Calgary/Project/OurSensor/Reporter
From 2013.igem.org
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<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 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> | + | <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> |
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| + | <p>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:</p> | ||
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Revision as of 02:45, 29 October 2013
Reporter
Reporter
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 enzyme/catalyst reporter 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.
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:
