Team:Thessaly/Proof Of Concept
Proof of Concept
Can our idea turn into reality?
Overview
This exceptional year, we built and characterized the monitoring system of Amalthea. The monitoring of the Gastrointestinal (GI) tract is accomplished by inserting a NOT-GATE device in our engineered bacteria, which detects the absence of Short-Chain Fatty Acids (SCFAs). SCFAs are diagnostic biomarkers for the evaluation of the function of gut flora, which is unbalanced mostly in IBDs. Through our Integrated Human Practices (Read more) we became aware of the need for a non-invasive evaluation of the gut flora and adjusted our project so an edible capsule hosting the engineered bacteria was designed.
To build the monitoring system, we designed and built three genetic modules, Tet-off, Prom, and GPCR-Tango, with scaled difficulty to increase the possibility of success. Each module contains a NOT-GATE device and a sensing element, which functions as an input. Explore our full design (Read more).
To demonstrate the proof of concept experiment for the detection and transmission of the desired signal, we used 2 Arduino UNO boards, a Bluetooth 433MHz link, and a potentiometer, since we lacked the required electrodes and solution. The potentiometer acts as a voltage divider, and the voltage it provides is captured by the Bluetooth Transmitter and passed on to the Receiver, just as it should happen with the SCFAs and the electrodes (Read more).
Prom Module
To demonstrate our system’s functionality, we chose to work with the Prom Module. This is because the Prom Module is promoter-based and thus has reduced complexity, while it is capable of SCFA detection. We assembled SCFA-inducible NOT-GATE devices and characterized them. To enable inhibition of the expression of eCFP we added 2mM of SCFAs, which showed optimal fluorescence during our characterization experiments Read more and our modeling Read more.
Figure 1. NOT-GATE-regulated eCFP fluorescence. The presence of acetate or propionate induces the activation of pFliC and then the expression of the reporter gene, eCFP is suppressed.
We measured eCFP fluorescence at 0, 4, 8, and 20 hours using a plate-reader in the presence or absence of SCFAs. Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates. The results of the assay can be seen in the figures below:
Figure 2. NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM acetate.
Figure 3. Cell growth in the presence or absence of 2mM acetate.
Figure 4. NOT-GATE-regulated eCFP fluorescence after in the presence or absence of 2mM propionate.
Figure 5. Cell growth in the presence or absence of 2mM propionate.
There is a slight increase in fluorescence in absence of SCFAs - acetate and propionate - as expected. We hypothesize that the gap will be bigger when adding butyrate, which is the main inducer for pFliC, and which we didn’t have in the lab as per Wiki Freeze date. As far as the cell growth goes, we expected a reduction of the cells, since we add acids that are toxic to the cells. These results indicate that our NOT-GATE device works, regarding these acids but in an expected range.
Transmission Module
Figure 6. The Arduino board on the left is connected to the Bluetooth transmitter (up).
First, a green wire starting from the 5V power supply pin of the UNO connects to a positive pin of the breadboard. The green wire then connects to the left pin of the potentiostat for power supply. A green wire extends from the left pin of the potentiostat and connects to the middle pin of the Bluetooth Transmitter for power supply. The orange wire starts from the ground pin of the UNO and connects to a negative pin of the breadboard. From there, the white wire connects to the right pin of the potentiostat to the Arduino ground. Another white wire starts from the right pin of the potentiostat and ends up on the left pin of the Bluetooth Transmitter, which is its ground pin. The potentiostat middle pin is connected to the A2 analog pin via the yellow wire, as it takes continuous values. This data passes on to the Bluetooth Transmitter via the blue wire, that connects the middle pin of the potentiostat to the right pin of the Transmitter.
The Bluetooth Receiver follows the same rules. A green wire from the UNO 5V supply pin connects to the leftmost pin of the Receiver (Vcc), while the orange wire connects to the rightmost pin of the Receiver(gnd). Both middle pins are interconnected and serve as data pins. They collect the output and pass it on back to the Arduino via the yellow wire, which is connected to the ~5 digital pin for output. From there, the output values can be viewed in the Arduino IDE.
