The programmable Smart Sleeve is the foundational unit on which the eVOLVER is built (Fig. 2 and Supplementary Fig. 1). The Smart Sleeve is comprised of all the sensors and actuators required to control the culture conditions inside a 40 mL borosilicate glass vial. At the core is an aluminum sleeve, which surrounds the vial and is used to control temperature via two resistive heaters and a thermistor integrated within. Near the base of the vial sits a 3D printed part that houses and aligns the optical density LED and photodiode. Below that sits a fan motor equipped with magnets to rotate a stir bar within the vial. The Smart Sleeve represents one of the most easily customized features of the eVOLVER: by changing which sensors and actuators are used and their layout, the user may develop culture vessels that fit their experimental needs. For a detailed description of the sensors and actuators used to control stirring, temperature, and optical density in Smart Sleeves featured in this study, as well as strategies for modifying the Smart Sleeve to fit experimental needs, refer to Supplementary Note 4. Liquid handling is also controlled at the level of the individual culture vessel, yet these components are housed in a separate fluidic module, described in Supplementary Note 5.
The sensors and actuators on each sleeve are integrated in a small printed circuit board (PCB), termed the Vial Board (VB). We designed the VB such that we can easily solder electrical connections and efficiently manage/package wiring from the sensors. The VB is a very simple PCB, containing only a few resistors, and is straightforward to redesign and inexpensive to manufacture, if needed. The simplicity in the VB leads to robustness in the system. For example, any accidental overflow and spillage from the vials (e.g. from clogged fluid lines or user error) should minimally impact the rest of the system, as critical components are located at the Motherboard rather than the sleeve itself. Ribbon cables provide a modular way to connect the integrated Smart Sleeves to the Motherboard.
The VB is designed to rest atop a 3D printed piece, which houses optical density and temperature components (see Supplementary Note 4). The printed part can be fabricated with Nature Biotechnology: doi:10.1038/nbt.4151 5 any commercial or DIY 3D printer, readily available at almost any university or hacker space, and customized to the requirements of the user. For example, if a user wanted to change the mode of optical density detection between scattering and absorption, they could redesign the 3D printed part housing the LED-diode pair such that it would have the correct offset angle for the desired mode of measurement.
Vial
Vial Holder
Vial Sleeve
Vial Cap
Vial Board
Based on previous work26, optical density measurements in a bioreactor can be measured with a simple 900 nm infrared (IR) LED and photodiode pair. There are two practical benefits of using 900 nm scattered light instead of the classic OD600. First, at 900 nm, turbidity/optical density measurements are less dependent on the absorbance spectrum of the media, meaning calibration is required less frequently before each experiment. Second, wavelengths in the visible range are preserved for light induction and colorimetric assays. To maximize scattering, the LED-diode pair is offset at a 135° angle. The 3D printed part is designed to house the LEDNature Biotechnology: doi:10.1038/nbt.4151 16 diode pair slightly above the height of the stir bar, at the correct angular offset. The part can be easily customized and printed to the users required specifications with any 3D printer. In the eVOLVER configuration used in this study, the IR LED and photodiode pair (4 leads) are each connected to the CMB via screw terminals in SA slots 4 and 5, respectively (Supplementary Fig. 7). In SA slot 4, a 16-channel PWM board amplifies a 3.3V signal from the Arduino microcontroller to a 5V signal to power the IR LED. A resistor is placed on the CMB to limit current and prevent the LED from burning out. SA slot 5 contains the 16-channel ADC board, responsible for analog filtering and demultiplexing the signal from the photodiodes. The ADC board reads the sensor by measuring the voltage difference across a 1M Ohm resistor, located on the Motherboard. Both slots are managed by Arduino 3 in the system developed in this manuscript. Briefly, the Arduino code interprets serial inputs from the Raspberry Pi, flashes ON the IR LEDs to measure turbidity, and responds with the current measurements. In the present system, optical density can be measured every 30 seconds, limited by the time taken for the Arduino to average diode readings (to minimize noise). For convenience, density readings from the 900 nm LED-diode pair were calibrated to OD600 measurements from a spectrophotometer, and the calibration curve fit with a sigmoidal function (Supplementary Fig. 8). Spectrophotometer readings were performed on a Spectramax M5 using 300 uL of media in a 96-well flat bottom plate; users may substitute density calibration data from measurements used in their labs. The optical density measurements in all experiments are calculated based on the calibration curve fit for each Smart Sleeve (Supplementary Fig. 8). For our experiments, calibration was performed using a dilution series of yeast cells suspended in distilled water, but in theory any cell type and/or solution of interest (such as evaporated milk) could be used. A custom MATLAB script was developed to facilitate the density calibration process, particularly important for bringing new eVOLVER units on line. Following calibration, the system was used to compare growth of S. cerevisiae (FL100) cells in eVOLVER vials to that in 250 mL flasks with 50 mL of media shaken at 300 rpm (Supplementary Fig. 8). Finally, to quantify the variance in growth across eVOLVER vials, 96 cultures across six 16-vial eVOLVER units were grown in parallel and aligned (Supplementary Fig. 8). These results demonstrate that eVOLVER cultures are repeatable, and exhibit comparable growth rates to cultures in shaken flasks. As previously mentioned, varying temperature induces a shift in the optical density readings (Supplementary Fig. 6). In measurements performed on yeast cells, we observed the largest shift near the center of the optical density calibration curve, while at low or high OD, the shift due to temperature was minimized. This information was used to select a density range for experiments in which temperature was controlled dynamically (see Fig. 4). As cells may shift in size in response to heating, we also quantified temperature-induced offset in optical density readings using evaporated milk.
The eVOLVER platform features tunable and independent stir rate control across culture vials. Stirring in eVOLVER is actuated by 12V brushless DC motors with attached neodymium magnets. The fastened magnets spin a stir bar (20 mm x 3 mm, PTFE coated) within an autoclaved glass vial (28 mm x 95 mm, borosilicate). The stirring module utilizes a single SA slot on the Motherboard; in the particular configuration described in this study, we utilized SA slot 1 (Supplementary Fig. 4). The two leads of the motor (12V & GND) are connected to a screw terminal on the component mount board, from which a ribbon cable connects the smart sleeve to the Motherboard. The PWM board (plugged into the SA slot) can control each motor independently to achieve different stir rates across eVOLVER vials. Briefly, the 16-channel PWM board amplifies a 3.3V signal from the Arduino microcontroller to a 12V signal to actuate Nature Biotechnology: doi:10.1038/nbt.4151 12 the motor. Arduino 1, which manages SA slot 1, was programmed to take in serial inputs from the Raspberry Pi and translate the serial values to different stir rates, determined by pulsing the motor ON and OFF at different ratios (Supplementary Fig. 4).
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.
Differences to Version C bolded
Fan
Unused; linked to fan for future fan tachometer
Heating resistor
Thermistor
OD IR LED
OD 135 photodiode
OD 90 photodiode / Spare A
Spare B
Differences to Version D bolded
Fan
Unused; linked to fan for future fan tachometer
Heating resistor
Thermistor
OD IR LED
OD 90 photodiode
OD 135 photodiode / Spare A
Spare B
The two photodiodes on the eVOLVER are at 90 degrees and 135 degrees from the IR LED.
Try OD135 for OD600 < 0.5
There will likely be vials that max out between OD600 0.5 and 1.0
Try OD90 for OD600 > 0.5
Some eVOLVER systems are able to read OD90 up to OD600 of 4 or 5
This may require optimization of the internal resistor pack
Switching between currently requires manual OD calibration (can be applied to an existing GUI calibration you have on your eVOLVER)
See this forum post for an example