Summary of Experimental Results
   1. Successful amplification of DNA template
          In order to test our RNA biosensors, we first needed to use PCR to amplify the gene from the plasmid template. We used purified plasmids containing our biosensor genes, primers that were complementary to the 5' and 3' ends of our biosensors, and a PCR kit to amplify the correct sequence. We then used Dpn1 to remove the original plasmid DNA and ran samples of the reaction mixture on a 3% agarose gel with the Promega 100 bp ladder. Unfortunately, some of the original plasmid remains uncut and can be seen as a band around 1000 bp but a prominent band can be seen for some of our biosensors at the right length of approximately 100 bp (Figure 1). Using Q5 DNA polymerase, we were able to produce DNA for the Glucose-1, Mango-1 and Scramble-1 constructs (Figure 1A). We then used Phusion DNA polymerase to attempt to produce the remaining two constructs (Figure 1B). Both the Glucose-2 and Theophylline-1 constructs were able to be amplified using this procedure.




   2. In vitro Transcription of RNA Biosensors
          The DNA templates created using PCR amplification all contained a T7 promoter upstream of the biosensor sequence. We used the Promega T7 RiboMax Expression kit (gift from Promega) to transcribe our RNA biosensors (Figure 2). Samples were taken at 15 min, four hours, and after DNase treatment to determine the progress and efficiency of the transcription reaction. All RNA biosensors were successfully transcribed as visualized by a 15% urea-PAGE and quantified using a BioDrop.




   3. Measuring Transcription Using RNA Mango
          RNA Mango has been used as a fluorescent tag for tracking small non-coding RNAs (Autour et al., 2018) and live cell imaging (Dolgosheina et al., 2014). Here, we used RNA Mango to look at transcription occurring in real time. The RNA Mango template DNA was mixed with the Promega T7 RiboMax Express kit and incubated with thiazole orange (TOI) in a cuvette. The fluorescence intensity was recorded using a Quanta Master 60 fluorescence spectrometer (Photon Technology International). TOI was excited at 510 nm and the emission spectra recorded between 520 and 600 nm. Readings were taken as soon as the DNA template was added to the reaction, then at 5, 10, 20, 30, 40, 50 and 60 minutes (Figure 3A). As time progressed, the fluorescence intensity increased and then reached a maximum and leveled off. Peak fluorescence at 535 nm was then plotted against time (Figure 3B). The data was then fitted with a logistic growth curve (y = a / (1 + b e-kx ), k > 0) and was found to have a doubling time of 0.1355 min (~8 sec).




   4. Glucose Aptamer Testing using RNA Mango as Biosensor
          In order to achieve tight regulation of insulin translation in our proposed system, we would need a glucose aptamer that could respond to biologically relevant glucose concentrations. To test our glucose aptamers we designed an in vitro assay that could report glucose binding based off the fluorescence intensity of a linked RNA Mango biosensor. Upon binding of glucose to its RNA aptamer, a conformational change within the RNA would result in more correctly folded RNA Mango and therefore higher fluorescence as it is now able to bind to the fluorophore thiazole orange (TOI). See our engineering page for more details (Engineering).



          From our experiments, we concluded that the addition of glucose to the RNA biosensor mix does result in an increase in fluorescence but only under certain conditions (Figure 4A). To use this biosensor to measure glucose binding, it first needed to be unfolded by heating to a high temperature and then allowed to refold and cool in the presence of glucose. The TOI was then added just prior to measuring the fluorescence. Our hypothesis is that the RNA is quite conformationally stable and cannot unfold to bind to glucose without first being melted. TOI can also not be added first, as it has a low nanomolar affinity for RNA Mango and would therefore bias the RNA folding to form the RNA Mango structure regardless of whether glucose was present or not.

          Additionally, the University of Manitoba team was able to test our RNA constructs (Figure 5). After addition of TOI, there seems to be a transition period for RNA biosensor to adapt to correct conformation to bind to TOI (which is shown through gradual increase in fluorescence). In the absence of glucose, it takes roughly 4-5 minutes to achieve maximum intensity. In the presence of glucose, it takes roughly 7-8 minutes to achieve maximum intensity. When evaluating the effect of glucose presence and absence, both have the same intensity at the start of the experiment. However, once maximum intensity is achieved, the presence of glucose leads to higher fluorescent intensity (Figure 5B and C).




References
Autour, A., C. Y. Jeng, S., D. Cawte, A. et al. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat Commun 9, 656 (2018). https://doi.org/10.1038/s41467-018-02993-8

Elena V. Dolgosheina, Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan, Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, Paul A. Wiggins, and Peter J. Unrau. ACS Chemical Biology 2014 9 (10), 2412-2420 DOI: 10.1021/cb500499x