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Our project aims to control the concentration of living bacteria in a culture. To do so, we designed a genetic network allowing light controlled cell growth. In addition, we built a device in order to send and receive light signals from the bacterial culture. We have thus to create a means of communication from cell to machine and from machine to cell. For cell to machine communication, we chose to measure the red fluorescence of KillerRed. The first function of our device is to excite and measure fluorescence intensity thanks to a light source, excitation/emission optics and a photodiode. In this way, our bacteria will be able to talk to our device. For machine to cell communication, we will use red light to activate light-inducible promoter that triggers KillerRed production and white light to generate ROS thanks to KillerRed phototoxic activity. In our system, the rate of KillerRed production and the number of living cells will be controlled by the intensity of the red and white light beams. Therefore, a second function of our device is to generate controlled light intensities at different wavelengths. In this way, our device will be able to talk to our bacteria.
Logical scheme of our device The computer gets information from the microcontroller about the fluorescence level of the bacterial suspension and calculates the intensity of the light to express KillerRed and control the cell density. The microcontroller itself controls the intensity and the spectrum of the light source that illuminates the sample. The photodiode measures the fluorescence level of KillerRed.
Our device is built in such a way that the user only needs to define the concentration of living cells he wants and put the Erlenmeyer with our engineered bacteria. From that moment on, the device works in standalone manner. It first measures the initial red fluorescence (the baseline). Then it induces the KillerRed protein using the red-inducible promoter. Every 5 minutes, Talk’E.Coli measures the level of red fluorescence of the culture and consequently of KillerRed. With these measurements and the parameters given by the model it can calculate the moment when it needs to activate the ROS emissions with white light to stabilize the living cell concentration. The main asset of the device is that even if there is a lower increase of the concentration of KillerRed or if the killing rate is higher since it measures every 5 minutes the red fluorescence it can adjust exactly the light intensity to correct these rates.
First, we will explain the choice of the different components, then the several experiments we did to find the most accurate parameters for each part of the device : the photodiode and Arduino, fluorescence measurement, the electronic circuit, the servomotor. All these elements were then integrated in the box that we designed and built.
