# Temperature

## Description

In contrast to current approaches in which all culture vessels are housed in a single incubator7,26, we developed a module for individually controlling the temperature of each Smart Sleeve in the eVOLVER. This not only allows the cultures to be maintained at distinct temperatures, but also reduces thermal mass, permitting dynamic temperature profiles. For the configuration described in this study, the temperature module utilizes SA slots 2 and 3 on the Motherboard (Supplementary Fig. 5). Typically, there are three main components to temperature control: (1) a thermometer, (2) a heater, and (3) a feedback controller. In our setup, the thermometer and the heater are integrated in the Smart Sleeve while the feedback controller is located on the Motherboard. Specifically, the temperature is measured by a 500 μm thick temperature-sensitive resistor, or thermistor (Semitec, 103JT-025). The sensor is integrated into the sleeve between the 3D printed part and the aluminum tube, and the thermistor is soldered onto the component mount board (CMB) after assembly. The aluminum tube enables even heat distribution/dissipation and shields the culture from ambient light (important for other measurements/parameters). Two heating resistors (20 Ohm 15 W, thick film) are screwed onto the aluminum tube for better contact and connected to the CMB via soldering. In our setup, the four leads, 2 from heating resistors and 2 from thermistor, are connected via a ribbon cable to the Motherboard and routed to SA slots 2 and 3, respectively. In slot 2, a 16-channel PWM board amplifies a 3.3V signal from the Arduino microcontroller to a 12V signal to actuate the heating resistors. Slot 3 contains a 16-channel ADC board, which reads the voltage difference across a 10 kilo Ohm resistor, and is responsible for analog filtering and demultiplexing the signal from the thermistor. These slots are connected to and are programmatically controlled by Arduino. Briefly, the Arduino code interprets serial inputs from the Raspberry Pi, updates the set point on the PID controller, and responds with the current measured temperature. Temperature settings can be updated as frequently as every 30 seconds. To determine how much to turn the resistive heaters on, the Arduino is programmed with a simple PID control algorithm. The PID controller can be easily tuned via software to obtain the desired overshoot and time delays. The Arduino then controls a PWM board (on SA slot 3) to interface with the resistive heaters and get the desired heat output. Calibration of the temperature measurement in the sleeve was performed by comparing the temperature of water measured in the vial using a thermocouple to the values returned by the thermistor (Supplementary Fig. 6). The dynamics of heating were determined by tracking temperature during a programmed step function, again comparing thermocouple and thermistor readings; the thermistor measures the temperature of the sleeve, while the thermocouple measured the actual water temperature. At room temperature (23°C for this experiment), a single culture (20 mL) can reach a temperature of 42°C in roughly half an hour with the current hardware setup (Supplementary Fig. 6). During an experiment, the transient offset between the recorded temperature and actual temperature may vary due to ambient temperature and volume of liquid. At steady state, the temperature can be maintained to +/- 0.1°C, with properly tuned PID constants. Max temperature and rate of temperature ramp can be changed with different power sources (e.g. 24V power source could reach temperatures >55°C). It should also be noted that at different temperatures, the optical density readings are affected accordingly. This effect was measured in both yeast cultures and evaporated milk (Supplementary Fig. 6). See the next supplementary note for more detail.

![Individually controllable temperature achieved by feedback between thermometer and heaters integrated in the Smart Sleeve. (a) Photographs of eVOLVER temperature components. A temperature-sensitive resistor, or thermistor, with a compact form factor, 25 mm x 3.6 mm (left). Sensor integrated into Smart Sleeve in between the 3D printed part and spray painted aluminum tube (center). Two heaters are screwed onto the aluminum piece and all components are soldered onto the CMB (right). (b) Schematic of system design for eVOLVER temperature module. The resistive heaters and thermistor are integrated into the Smart Sleeve and interface with PWM and ADC boards at SA slots 2 and 3, respectively. Arduino 2 manages both boards and interprets the desired temperature settings and responds with the current temperature (right). The temperature is maintained with a PID controller programmed into the Arduino. The controller interprets the input from the ADC board in slot 3 to determine the output of the PWM board in slot 2.](https://3394537773-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FYy5AdmKzUTn7D8n357Md%2Fuploads%2FiAX0jRg3NzQu8CW8buMD%2Fimage.png?alt=media\&token=eb1542b1-863b-4295-ae4a-82dc8d0b6093)

![Temperature control characteristics in eVOLVER Smart Sleeves. (a) Temperature calibration curves. Top: A thermocouple was used to measure the temperature at different thermistor readings. The points were fit with a line and all temperature measurements in the experiment were calculated based on the fitted line. Bottom: Recalibration, after a year of use, demonstrates stable and robust temperature control in Smart Sleeves. (b) Temperature offset between aluminum sleeve and liquid. To measure the temperature offset during dynamic temperature changes, the integrated thermistor (upper left) and a thermocouple (lower left) simultaneously recorded temperature at two different locations during a square wave (right). (c) Impact of temperature changes on optical density readings. Optical density calibration curves for yeast cultures (see Supplementary Fig. 8) were generated at three different temperatures, and verified separately by OD600 spectrophotometer readings (left). To characterize temperature-induced OD offset without cells, evaporated milk was used to generate another set of calibration curves at different temperatures (right).](https://3394537773-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FYy5AdmKzUTn7D8n357Md%2Fuploads%2FMpnJ47oRtOV8IoCnetgH%2Fimage.png?alt=media\&token=a9c55728-1ad6-439c-bd22-715503dfdff2)
