• Pump 1: Used to pump out medium from the reactor bottle into the OD-chamber and finally to the waste bottle
• Pump 2: Used to dilute the reactor medium with fresh sterile medium, if required
• Pump 3: Used to pump out reactor medium through the heating block and the fluorescence chamber into another waste bottle
• Arduino 1: Arduino controlling the H-Bridge module that had the peristaltic pumps attached
• Arduino 2: Arduino running the OD-measuring code, sending GPIO signals to Arduino 1, if necessary

Arduino 2 was used to run the "Aachen final.ino", an Arduino code used by the 2015 iGEM team Aachen to measure the optical density of a medium with the "TSL 235R-sensor". Before use, the system had to be calibrated with samples of a known OD (measured with a laboratory OD-device). The output value was transformed into real OD-values for better control of the experiment. Then a "switch case" code was written using this output value to decide whether fresh medium was needed to dilute the reactor medium or if a defined pause was required for the next measuring cycle. The Arduino 2 communicated with Arduino 1 via GPIO signals.

For a measuring cycle the coding on the Arduino 1 was designed as follow: After 20 minutes had passed, a small amount of medium was pumped out of the reactor and through the OD-chamber from below. Then a 5 second delay was used for any bubbles inside the chamber to dissociate. A delay ensured Arduino 2 to measure the optical density in the meantime. Then the code was waiting for GPIO signals of Arduino 2 that triggered the pump 2 if necessary. Our experiments showed that the optimal optical density for proper fluorescence signals was at an OD₆₀₀ of 0.3. The OD-maintenance system was set to this value and was able to keep the OD₆₀₀ at ~ 0.3 over a long period of time (or until there was no fresh medium left). Whenever an OD₆₀₀ bigger than 0.3 was reached, approximately 5 ml of fresh sterile medium was pumped into the reactor bottle.

One problem that came up was that the liquid level inside the reactor bottle - it should stay the constant over longer periods of time. Since liquid was pumped out of the reactor bottle every 20 minutes for OD and fluorescence measurements by the other pump, the liquid levels dropped relatively fast and the reactor bottle was empty pretty quick in first trials. The solution was to measure the volumes transferred by the peristaltic pumps for specific running times. Then it was possible to adjust the liquid level inside the reactor bottle by changing the Arduino code since the estimated volumes were known. Every pumped out volume of reactor medium was replaced with the same amount of fresh sterile medium after this adjustment.

Thermostat including a heating mat and a temperature sensor - for the maintenance of the optimal temperature inside the reactor bottle, a basic thermostat system was built:

• Arduino nano: Running a temperature measuring code (DallasTemperature.h and OneWire.h)
• DS18B20 digital temperature sensor: Placed inside a glass tube that was inserted inside the reactor bottle and filled with distilled water. The glass tube was built by melting the opening of a glass burette over a bunsen burner until it was closed and air-tight
• 12W heating mat: Stuck on a small piece of aluminium and placed beneath the reactor bottle
• Relay module: Suited for common microcontrollers

The relay module and the heating mat was connected to a 12V energy source. The controller pins of the relay module were triggered by the arduino nano, whenever the inside temperature of the reactor medium went higher than 30°C. This caused the relay module to stop the energy supply to the heating mat. Several test runs showed that this set-up holds the inside temperature between 29°C and 30°C which was sufficient for our purposes.