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Interview with Dr. Keith Pardee
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We read your paper from last year titled “Rapid, Low-Cost Detection of Zika Virus Using
Programmable Biomolecular Components” and we wanted to know, what led you to
use β-galactosidase as a colored readout?
Dr. Pardee: At that time, we hadn’t considered using C2C2 (Cas13a). So, one of the reasons was
because of the enzyme activity. Single molecule of reporter could serve amplification, rather than
just fluorescence. The other (reason) was just for practical use. No need (to use) UV lights,
electronics, camera, and could be done in a simple piece of paper. Tried 10 different enzyme
options with other based reporters.
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What was your motivation to build that detector on your study?
Dr. Pardee: Multiplexing, being able to track multiple reactions at the same time, and the
potential for quantification. These processes are semi-quantitative and so with that information,
you want to be able to read rather than just being plus minus, being able to calibrate your
reactions. You might be not being able to say exactly how much you have but you may be able to
say this is a high titer or a low titer individual. Also, user accessibility, if you can automatize some
of this tests for reading like in pregnancy tests with a digital detector… can make it really easier
to use.
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Regarding our project
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What do you think about the problem of antibiotic resistance and the ways to prevent
it?
Dr. Pardee: This is probably the biggest question and it is not my area of expertise. We should
continue the advocacy of responsible use in agriculture and in patients. With tools that we both
are building where we can identify resistance early and contain it and treat individuals that have
resistance is also going to be important.
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We have some problems getting the Cas13a lyophilized into paper. Do you have any
low-cost suggestion for us?
Dr. Pardee: I have never done that but there was a new paper published (Kikuta et al., 2017) that
uses trehalose as a cryoprotectant, that lets you dry cell free-reactions at room temperature. The
trehalose is present in water bears and seeds, and helps keeping things dry. It is an inhibitor of
cell free-reactions at high concentrations.
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Did you ever considered using as a colorimetric readout such as gold nanoparticles?
Dr. Pardee: Yes absolutely. I think is a great thing to do and useful. You can borrow so much from
what is already been done.
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Michael and Julian from the iGEM BOKU Vienna came to do some experiments in our lab on 4th and 5th of October. Their iGEM project is called D.I.V.E.R.T. (Directed in vivo evolution via reverse transcription) and they are trying out new strategies for in vivo evolution which shows potential advantages over classical in vitro methods. For this they use yeast and E.coli to demonstrate their concept. They used the flow cytometer in our lab in Garching to better characterize their constructs from E.coli and S. cerevisiae. For the use one of our lab member explained them how to use the flow cytometer in the lab and also provided them the necessary help. It was a long day work for them but they got convincing results by the end. We also had a small gathering in the evening before they left together with the old igemers who came to meet us. We were very happy to have them in our lab and to get to know each other's team.
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