Fluorimetry1. Fluorimetry in bulk culture shows oscillatory behavior and characteristic burst
We characterized the circuit using the spectramax M2e spectrofluorometer, using the protocol described in the characterisation section of the project tab. Samples taken every 10 minutes were checked for fluorescence by excitation at 585 nm and emission at 604 nm was recorded. The results were plotted in R in a 3-D plot for the fluorescence at different OD (cell concentration) and time values. We then ran a support vector regression algorithm to find the values of fluorescence at a constant OD value. We chose to plot the fluorescence at the root mean square OD value, in order to best account for and normalize the results for variations in the OD value throughout the experiment.
We were able to capture the oscillatory behavior of the circuit in a time course of ~150 minutes. Plotted against the negative and positive control (figure 1), we see our iDanino circuit showing a sudden burst of fluorescence at a time of around 150 minutes, which is similar to what has been reported by Danino et al (nature, 2011). This shows that our system seems to be in an oscillatory behavior in bulk, indicating the working of the circuit as expected.2. Characterising the spike in the oscillations of the iDanino circuit
The spike in the RFU levels of the circuit is apparent from the figure. We further calculated the max/min ratio and max – min values of all the three samples, which is indicative of the gain in amplitude of the values (table 1). We see that the highest value of max/min ratio (relative gain) and max – min (absolute gain) values obtained are for the iDanino circuit, indicating a solid gain in oscillations.3. Results of the fluorimetry of iDanino circuit is in accordance with the Danino circuit fluorimetry values
We ordered the plasmids pTD103_aiiA and pTD103_LuxI_sfGFP, constructed originally by Tal Danino, from addgene, and ran the same experiment for these plasmids, which we used as a further proof of the working of our circuit (since the pTD plasmids were confirmed to be working). Running the fluorimetry experiment for the pTD plasmid containing cultures (positive control, negative control, and the complete circuit), plotting fluorescence at the root mean square value of cell concentration after support vector regression based curve fitting. We saw a similar spike in the OD values in this case as we saw for our own iDanino cultures, indicating that our oscillator worked as expected.