Team:Northwestern/Interviews
Project significance and design
Interview with Dr. Jewett
To better understand how certain design aspects would affect both the public’s willingness to use our sensor as well as the quality of the sensor, we interviewed Professor Michael Jewett, Chemical Engineering PhD.
In the initial MetaSense design, we planned on using a GFP output to indicate the presence of lead or chromium. However, when talking to Professor Jewett about this, he suggested that we switch to a colorimetric output for many reasons. One of such was that using a GFP reporter would require the use of a tool that can measure fluorescence - something that few citizens have access to. Thus, MetaSense users would be required to send their paper-based assay to a laboratory in order to receive results, which would take time and resources. Professor Jewett instead suggested that we make use of a colorimetric output to indicate the presence of the specified heavy metal. The reason for this modification is that it will ideally provide a readout in around 2 hours, while also not requiring users to send their sensor into a laboratory.
Additionally, our initial design only called for one plasmid. While talking to Professor Jewett, he suggested that we make use of a two plasmid system. One plasmid would consist of a constitutive promoter, followed by a gene which codes for a repressor protein that is inactivated by either lead or chromium. The other plasmid would contain an operator sequence to which the aforementioned repressor protein could bind, followed by a gene coding for an enzyme that produces a colorimetric output in the presence of a certain substrate. As a result, we would be able to tune the concentrations of these two plasmids to enhance the accuracy that MetaSense provides regarding heavy metal concentration.
Finally, Michael Jewett connected us with a graduate student in his lab, Adam Silverman, who focuses on cell-free sensing - a resource that proved integral to much of our progress this summer.
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Interview with Adam Silverman
In order to better understand cell-free sensing, we interviewed Adam Silverman, a Chemical and Biological Engineering PhD student. During the interview, we went over two cell-free sensing systems that Adam had worked with.
The first system we talked about was a two-plasmid repressor system which produces β-galactosidase in the presence of DAPG. This system is optimized for cell free expression, so we decided that we should utilize the plasmid backbones in MetaSense. To do this, we simply would need to amplify the backbones using PCR while also taking out the promoters and reporter gene. Then, using Gibson assembly, we would be able to clone in promoters that contain a binding site for chromium/lead regulated repressor proteins, as well as an output gene.
To first show that these backbones are optimized for cell-free protein synthesis, Adam suggested that we adapt the plasmid backbones to sense AHL. This system is similar to the DAPG sensor, except that it produces an activator protein and the presence of AHL is required for the activator to bind to the promoter, resulting in a GFP output. Again, to create this system we need to clone in the pLux promoter and LuxR repressor gene into the backbones of the DAPG sensor.
Ultimately, Adam provided crucial insights into cell-free protein synthesis and certain aspects that affect its quality. Given these insights, we adapted MetaSense to include a two-plasmid system that will provide better results than other systems, thus improving the quality of our design.
Interview with Dr. Alam
To finalize our project idea as well as go over some design specifications, we interviewed Khalid Alam, a postdoc at Northwestern University.
Initially, we talked through our ideas for possible projects, and he suggested that we go forward with hexavalent chromium and lead sensing. The reason for this is that it has a local impact; hexavalent chromium and lead levels in Evanston, as well as the greater Chicago area, are above the EPA deemed limits. Additionally, U.S. Steel has contributed significantly to this issue through their dumping of a large amount of hexavalent chromium into Lake Michigan.
After deciding to focus on chromium and lead sensing, we started to define our end goal. One of the main aspects that we wanted our project to incorporate is citizen science. To do this, we must design a sensor that could be distributed for use by citizens. As a result, we decided that our end goal should be a paper-based sensor because it will allow for citizens to easily test their water, thus increasing their involvement in citizen science. The paper-based nature of our product requires that we work in cell-free, which offers many advantages over in vivo sensing. One such advantage is that while working in cell-free, we don’t need to worry about keeping cells alive while also trying to sense lead and chromium. Instead, we just need to supply the engineered DNA, along with the reagents necessary for transcription and translation. Another advantage that results from this is that we are able to control the concentration of DNA in the reaction, which will make tuning our biosensor easier as opposed to if it were in vivo.
Overall, our meeting with Dr. Alam proved crucial in defining our project, as well as determining certain aspects of our design that would increase its relevance to citizens.