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.

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 horseradish peroxidase. 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 E. coli. 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. 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 two candidates: the ampicillin resistance enzyme beta-lactamase and ferritin, 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 flexibility in our platform technology to meet the different criteria that will 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:

Prussian-Blue Ferritin

Beta-Lactamase