Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Summary

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Abstract

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

Introduction

Optogenetic systems use light to control a growing list of cellular processes including gene expression,^1,2,3,4,5 protein localization,⁶ protein activity,^6,7,8 protein binding,^8,9,10 and protein degradation.¹¹ A method for culturing cells in a controlled environment with programmed optical stimulation and for measuring their response over biologically relevant timescales is necessary to exploit the potential of these tools for research in cell biology and biotechnology. Our method takes advantage of chemostasis to maintain a constant cell growth rate in a well-mixed, aerated, and temperature-controlled glass culturing vessel^12,13 that is exposed to programmed illumination. We image individual cells in the culture effluent with an inverted microscope to measure the response of the culture to programmed illumination. The culturing, sampling, imaging, and image analysis are fully automated so that the fluorescence intensity and cellular morphology of the effluent cell culture can be measured over multiple days without user input.

This protocol can be implemented in most labs familiar with growing cell culture and microscopy, and the apparatus used is inexpensive and made of readily available components. A transparent culturing vessel is placed above a matrix of light emitting diodes (LEDs) capable of emitting 1 µW/cm²-10 mW/cm² of light. Microbes are grown in the culturing vessel continuously; one peristaltic pump is used to add media at the dilution rate, another is used to withdraw culture at a lesser rate to the microscope, and the difference escapes through an overflow outlet. A heating pad maintains the temperature. Air is continually pumped into the culturing vessel to maintain a positive pressure as well as to mix and aerate the culture. Except for the air pump, power to these devices is regulated by a microcontroller that also receives input from a thermometer and a connected desktop computer. The effluent cell culture is pumped to a microfluidic device on the stage of an inverted microscope. Non-fluorescent and fluorescent images are automatically acquired. The cells in the images are characterized by an algorithm that locates each cell as a region of interest (ROI) and measures the properties of each ROI.

To demonstrate an application of this protocol, we measured the response to varying light intensities of Saccharomyces cerevisiae cells engineered with a blue-light responsive optogenetic system which controls the transcription of fluorescent protein. S. cerevisiae, commonly known as baker's yeast, was selected because multiple optogenetic systems for controlling gene expression in this system already exist^14,15,16. Furthermore, this model organism is commonly used for studies in systems biology¹⁷ and as a chassis for biotechnological applications^18,19,20. Our representative results demonstrate that this protocol can be used to control transcription of a culture over multiple days by varying input light intensities and measuring the production of a fluorescent reporter.

Protocol

Figure 1: The continuous culturing apparatus. This simplified diagram shows how the apparatus should be assembled when it is used to culture, illuminate, and measure optical properties of the microbes. Please click here to view a larger version of this figure.

Figure 2: Overview of the protocol. The steps in the shaded region must be repeated every time the protocol is used. Closed loop control is possible³⁴, but is not implemented in this protocol.

1. Assemble the Thermometer to the Circuit Board

Figure 3: Connections to read thermometer values. This diagram shows how the digital thermometer should be connected to the PCB so that the microcontroller can get feedback to control the temperature of the culture. Please click here to view a larger version of this figure.

1. Solder the Ground, Data, and positive Voltage lines of the digital thermometer to their respective through-holes marked "G D +V."
2. Clip off one pin from a female 3-pin header and trim the remaining 2 pins with wire clippers, such that it can fit into the two through-holes labelled "R2" and not obstruct the microcontroller. Solder this in place. Connect the two soldered pins by inserting a 4.7 kΩ resistor in the pin header.

2. Connect the Power Control Components to the Circuit Board

Figure 4: Connections to control power to the heating pad. This diagram shows how the PCB and accessory parts should be assembled in order to control power to the heating pad. The parts to control the peristaltic pumps are connected in a similar manner. Note that the components for the thermometer have been removed for clarity. Please click here to view a larger version of this figure.

1. Trim off 1 cm from the pins of five female 3-pin headers. Solder these to the positions on the printed circuit board (PCB) marked as "#1," "#2," "#3," "#4," and "C B E."
2. Trim off 1 cm from the pins of a female 6-pin and 8-pin header. Solder these side by side to the column of through-holes labeled K1 through K14.
3. Connect the heating pad to the circuit board.
   1. Insert the ends of a 100 kΩ resistor in the through-holes labeled K1 and K2 on the PCB. Insert a metal-oxide-semiconductor field-effect transistor (MOSFET) into the position marked #1 on the PCB, with the label of the MOSFET facing towards the K1 through K14 through-holes so that the source pin is closest to the "#" label, and the drain pin is furthest from the "#" label.
   2. Cut the ground line of the 5 V direct current (DC) power supply cable and connect it to J2. Connect the ground line of the heating pad to J1. Connect K3 to pin 3 with a 1 kΩ resistor. Current should now only flow from J1 to J2 when pin 3 is set to +5V.z
      NOTE: The resistor/MOSFET set functions as a switch that allows current to flow from pin J1 to J2 when pin 3 of the microcontroller is set to +5 V.
4. Connect the slow peristaltic pump.
   1. Insert the ends of a 100 kΩ resistor in the through-holes labeled K4 and K5 on the PCB. Insert a MOSFET into the position marked #2 on the PCB, with the label of the MOSFET facing towards the K1 through K14 through-holes so that the source pin is closest to the "#" label, and the drain pin is furthest from the "#" label.
   2. Cut the ground line of the 12 V DC power supply cable of the slow peristaltic pump. Connect the peristaltic pump facing end to J3 and the wall wart facing end to J4. Connect K6 to pin 4 with a 1 kΩ resistor. Current should now only flow from J3 to J4 when pin 4 is set to +5 V.
5. Connect the fast peristaltic pump
   1. Insert the ends of a 100 kΩ resistor in the through-holes labeled K7 and K9 on the PCB. Insert a MOSFET into the position marked #3 on the PCB, with the label of the MOSFET facing towards the K1 through K14 through-holes so that the source pin is closest to the "#" label, and the drain pin is furthest from the "#" label.
   2. Cut the ground line of the 12 V DC power supply cable of the fast peristaltic pump. Connect the peristaltic pump facing end to J5 and the wall wart facing end to J6. Connect K9 to pin 5 with a 1 kΩ resistor. Current should now only flow from J5 to J6 when pin 5 is set to +5 V.

3. Connect the LED Matrix to the Circuit Board

Figure 5: Connections to control the LED matrix. This diagram shows how the LED matrix should be connected to the PCB. It also shows that the PCB can be stacked on the microcontroller. Note that the components for the thermometer and for controlling DC power to other devices have been removed for clarity. Please click here to view a larger version of this figure.

1. Solder the 6-pin, 10-pin, and the two 8-pin female pin headers into the through-holes on the sides of the PCB such that it can be stacked on top of the microcontroller.
2. Solder the 100 nF and the 10 µF capacitors to their marked positions on the PCB, noting that the negative terminal of the 10 µF capacitor (shorter lead) should be connected to the through-hole marked with a negative sign.
3. Solder the LED driver to the 2 by 12 set of through-holes, with the indentation on the driver far from the through-holes reserved for the LED matrix.
4. Solder 2 columns of male pin headers to the through-holes marked for the LED matrix, and trim the ends of these underneath the breadboard such that they will not obstruct the microcontroller. Connect these to two 8-wire strips of female/female jumper wires. Connect a second set of male pin headers to the other end of the jumper wires.
5. Insert the LED matrix over the median of the breadboard, and then insert the second set of pin headers to the columns on either side of the matrix. Ensure that the electrical connections are the same as if the LED matrix had been directly connected to the PCB with the labeled side of the matrix corresponding to the labeled column of through-holes.
6. Clip off one pin from a female 3-pin header and trim the remaining 2 pins with wire clippers, such that it can fit into the two through-holes labelled "R1" and not obstruct the microcontroller. Solder this. Connect the two soldered pins by inserting a 1 kΩ resistor in the pin header.
   Note: The culturing vessel will be stacked over the LED matrix. If the wire ribbons obstruct the vessel, offset them from the matrix. Then, use solid core wire to bridge the gap between the matrix pins and the wire ribbon pins, such that the electrical connections do not change.

4. Install Software and Connect to Hardware

1. Stack the PCB on the microcontroller.
2. Follow the links in the materials list to download the Integrated Development Environment (IDE) and custom code for the microcontroller.
3. Connect the microcontroller to the microscope computer via an A-B universal serial bus (USB) cable. Compile and upload the custom code to the microcontroller.
4. Download Micro-Manager^21,22 and FIJI²³. Configure Micro-Manager as a FIJI plugin by copying all ".dll" files, the "mmplugins" directory, and the "mmautofocus" directory from the directory where Micro-Manager was downloaded into the "Fiji.app" directory. Also, copy the "plugins/Micro-Manager" directory into the "Fiji.app/plugins" directory.
5. Download the "BioreactorController.jar" file into the "Fiji.app/mmplugins" directory.
6. Open Micro-Manager from FIJI> Plugins> Micro-Manager> Micro-Manager Studio. Use the Hardware Configuration Wizard to configure the software to control the microscope. Include the "FreeSerialPort" device in the Wizard with the label of the port to which the A-B USB cable is connected.

5. Make and Characterize the Light-proof Enclosure for the Culturing Vessel

1. Stack three 8 pin IC sockets as building blocks on the breadboard at each corner of the LED matrix such that the culturing vessel can rest on them above the matrix.
2. Fasten a portion of diffusion paper under the top layer of the sockets, such that the light striking the culturing vessel from the LED matrix is diffuse.
3. Cut out three 8" by 6" portions of black foam. Cut out a portion that is the same size as the electronic breadboard (2.3" x 3.5") from the inside of the first two and a portion that is the size of the rectangle made by the IC sockets (0.9" x 1.8") from the inside of the third. Stack these layers over the LED matrix such that the final layer containing the 0.9" x 1.8" aperture lies even with the top of the IC sockets, and surrounds the LED matrix.
4. Cut an additional sheet of black foam in half to make a 6" by 24" rectangle. Roll this into a hollow column of black foam that the culturing vessel can fit into with room for tubes and wires, such that it will thermally and optically insulate the culturing vessel.
5. Center the hollow column of black foam over the LED matrix, and mark the boundary of the column on the layer of black foam under it. This boundary will mark where it should be centered in the future.
6. Cut out a 3" x 3" portion of black foam. Use this later as a lid that can be taped to the top of the column to block external light.
7. Attach the photodiode power sensor to the culturing vessel's cap with tape, attach the cap, and insert the culturing vessel in the enclosure.
8. In Micro-Manager, go to Plugins> Bioreactor Controller. Set the matrix to illuminate at a subset of the range of possible intensities at 30 s intervals.
9. Record the light intensities as displayed on the power meter console connected to the power sensor. These measurements will enable the actual intensity of light to be known when the number of LEDs that are lit and the pulse-width-modulated (PWM) current to those LEDs is set.

6. Prepare the Culturing Vessel

Figure 6: Vessel connections. This diagram shows how the vessel and tubing of the apparatus should be connected prior to being autoclaved. Please click here to view a larger version of this figure.

1. Mark the height corresponding to every 2 mL increment of liquid in the range of 10 mL to 30 mL in the culturing vessel. Insert 10 mL of sterile deionized water, mark the water level, add 2 mL, mark the water level, and repeat until the 30 mL level is marked. Cover the markings with clear tape so that they are not easily removed, and dispose of the liquid.
2. Place the long end of the aluminum port through the silicone gasket, into the culturing vessel. Screw on the cap.
3. Cover one short aluminum port outlet with 1/16" ID silicon tubing. Plug the distal end by inserting a female luer lock and then connecting a male luer lock plug. This outlet is supplementary, and will not be used (tube 6 in Figure 6).
4. Connect two short segments of 1/16" ID silicone tubing with male and female luer locks. Connect one end to a short aluminum port outlet and plug the distal end with a male luer and female luer lock plug. The vessel will be inoculated through this tube (tube 7/7.1 in Figure 6).
5. Connect two 1/16" ID tubes to the ends of the 1/16" ID peristaltic tubing and connectors. Connect one end to a short aluminum port and plug the other (tube 4 in Figure 6). Later, this will be connected to the media flask.

7. Prepare the Media Flask

1. Connect a 1/16" inner diameter (ID) silicone tube to the longest aluminum port. The tube should be long enough to reach the media flask. Connect a 1/8" ID male luer lock to a short segment of 1/16" ID tubing, connect this to the preceding tube,, and then insert this short segment into the rubber stopper on the media flask (tube 3 in Figure 6).
2. Clamp the tube. This connection allows air to flow from the media flask to the culture vessel. When the media flask is later pumped with air and this tube is unclamped, the culture will be mixed, aerated, and kept at a positive pressure by the incoming bubbles.
3. Insert a 1/16" ID tube long enough to reach the bottom of the flask into the stopper, through which media will be transferred. Connect another 1/16" ID tube to this one, and then a 3/16" ID tube to that. Plug this with a 3/16" ID female luer lock and a male luer lock plug (tube 1/1.1 in Figure 6).
4. The third hole in the stopper should be filled with a short segment of 1/16" ID tubing, connected to two other segments of medium diameter tubing and plugged at the distal end (tube 2/2.1 in Figure 6). This will be connected to a vacuum pump and later to an aquarium pump.
   NOTE: If the tubing cannot easily fit through the holes in the rubber stopper, cut the ends of the tubing at a slant. Then, the slanted end which is pushed through the hole can be used to pull the rest through.

8. Prepare the Effluent Flask

1. Insert a male luer lock into a segment of 1/16" ID silicone tubing, and then firmly insert 0.022" ID polytetrafluoroethylene (PTFE) tubing. Separately, attach a female luer lock to a 1/16" ID tube. Then, thread one end along the PTFE tube to connect the locks and the other to a short aluminum port (tube 5 in Figure 6).
2. Connect the exposed 1/50" ID silicone tube to the 1/50" ID peristaltic tube and connectors. To this, connect three segments of 1/50" ID tubing and two segments of 0.022" ID PTFE tubing, alternating them. Connect this to the effluent flask via 1/16" ID tubing (tube 5 in Figure 6).
3. Connect one end of a 1/16" ID silicone tube to the second longest tube of the aluminum port and the other end to the effluent flask. This aluminum tube sets the culture volume, and excess culture overflows into the effluent container (tube 8 in Figure 6).
4. Autoclave the culturing assembly at 121 °C and 15 psi (general sterilization) for 30 min.
   Note: The tubes through which the media flask is filled, through which the media flask is vacuumed, and through which the culturing vessel is inoculated have multiple segments of tubing so that if the distal segment is contaminated, it can be removed to reveal a sterile segment.

9. Prepare the Microfluidic Channel

1. Mix polydimethylsiloxane (PDMS) and curing agent in a 9:1 ratio. Degas and pour onto the silicon master mold.
2. Cure the PDMS for 2 h at 65 °C and then cut it from the mask with a razor blade. Cut around the PDMS until it releases from the mold. Avoid pushing down and breaking the fragile wafer.
3. Use a 1.2 mm ID biopsy hole puncher to punch holes at both ends of the channel.
4. Plasma bond the PDMS to the cover glass²⁴.
5. Tape the long ends of the cover glass to the supporting aluminum frame such that the PDMS channel is centered on the aluminum frame, and then bake the PDMS again for 2 h at 65 °C.

10. Fill the Media Flask

Figure 7: Adding media. This diagram shows how media should be vacuum filtered into the media flask. It ensures that the media remains sterile. Please click here to view a larger version of this figure.

1. Make appropriate media. If the continuous culturing system will be operated as a chemostat, use media limited for a specific nutrient.
   NOTE: Examples of standard media composition for studies with S. cerevisiae have been previously published^{25,26,27,28,29}.
2. Attach the vacuum filter to a 100 ml bottle, remove the nipple cover, then remove the white plug from the nipple of the vacuum filter with sterile tweezers.
3. Connect the 3/16" ID silicone tube to the nipple, the media flask's other free tube to a vacuum pump (tubes 1 and 2 in Figure 7, respectively), and ensure that the tube connecting the media flask to the culturing vessel is clamped shut (tube 3 in Figure 7).
4. Fill the filter with media, turn on the vacuum pump, and then filter the rest of the media. Clamp the 1/16" ID silicone tubing connected to the vacuum (tube 2 in Figure 7) and turn off the vacuum pump.
5. Remove the leurs from the intermediate segment of 1/16" ID tubing connected to the vacuum pump so that an uncontaminated end of the tube is accessible. Insert the blue end of a sterile syringe air filter into this tube.
6. Clamp the 3/16" ID silicone tubing (tube 1 in Figure 7) and disconnect the male luer from it. Disconnect the plug from the female luer of the culturing vessel's media inlet tube. Connect these male and female luers to enable media to be pumped into the culturing vessel.
7. Remove the vacuum filter and cap the 100 mL bottle of media which was attached to the vacuum filter.
8. A day before the culturing vessel will be inoculated, inoculate a single colony of the optogenetic microbe in a test tube with 4 mL of the media collected in the previous step, and let it grow overnight at 30 °C or the culture's optimal temperature growth temperature with agitation.

11. Assemble the Apparatus Around the Microscope

1. Set the continuous culturing assembly near the microscope with the media flask higher than the culturing vessel and the effluent flask lower than the culturing vessel, such that the tube composed of segments of 0.022" ID PTFE tubing (tube 5 in Figure 6) can reach the microscope stage. Securely tape down the rubber stoppers on the media flask and effluent container.
2. Unplug the ends of the 0.022" ID PTFE tubing from the 1/50" ID silicone tube connecting them (tube 5 in Figure 6), and plug these ends into the inlet and outlet of the microfluidic device.
3. Connect the white end of the syringe air filter to the aquarium pump. The air pressure will push media into the culturing vessel.
4. When the media reaches the level of the effluent port, wrap the 1/16" ID media inlet tubing and connectors around the slow peristaltic pump (tube 4 in Figure 6) and the 1/50" ID sample outlet tubing and connectors around the fast peristaltic pump (tube 5 in Figure 6).
5. Aerate the media by unclamping the air tube between the media flask and culturing vessel.
6. Tape the heating pad and thermometer to the culturing vessel so that its temperature can be controlled. Coil the media tubing around the culturing vessel so the entering media will be at the same temperature as the vessel.
   NOTE: The PWM current to the heating pad is regulated by the microcontroller which uses inputs from the thermometer to keep the culturing vessel at its setpoint temperature.
7. Insert the culturing vessel into the black foam enclosure over the LED matrix, and ensure that the tubes are not pinched.
8. In Micro-Manager, go to Plugins> Bioreactor Controller. Set the "Media pump ratio on" field to 0.1 and the "Sample pump ratio on" field to 0. The flow rate will be very low, but greater than the rate of evaporation.
9. Clamp the incoming air tube between the media flask and culturing vessel. Remove the plug from the inoculation tube (tube 7 in Figure 6), and inoculate the culturing vessel with 1 mL of starter culture via a serological pipette. Clamp and plug the distal end of this tube to ensure that contaminants do not enter the culturing vessel, then unclamp the incoming air tube.
10. Cover the enclosure so that no light enters, and let the culture grow overnight.

12. Calibrate the Pumping Rates

1. In Micro-Manager, go to Plugins> Bioreactor Controller. Set the "Media pump ratio on" field to 0.5 and the "Sample pump ratio on" field to 0.
2. Disconnect the overflow tube from the effluent flask (tube 8 in Figure 6) and the sampling tube from the effluent flask (tube 8 in Figure 6) and collect effluent in separate vessels. Collect effluent for 1 hr, beginning after the pumps have been on for 15 min.
3. Calculate the flow rate of media into the culturing vessel from the volume collected in the vessel as:
   
4. Adjust the value of the "Media pump ratio on" field by this linear estimate:
   
   where
   
5. Iterate through this calibration procedure until the difference between the desired flow rate and the measured flow rate is < 0.2 mL/h, where the flow rate is averaged over a 1 h period.
6. Increase the value of the "sample ratio on" field and calibrate it in a similar manner until roughly 4/5^th of the volume leaving the culturing vessel is pumped out by the sampling pump and roughly 1/5^th of the volume is leaving through the overflow port.
7. Let the culture density in the continuous culturing apparatus equilibrate under these conditions overnight.
   NOTE: If the target flow rates cannot be reached, change the peristaltic pump tubing. Liquid is pumped at a higher rate when larger diameter tubing is used. Follow step 14 and then return to step 8.4.

13. Collect Microscope Images of Cultured Microbes

1. Fill the Stage Position List in Micro-Manager with a set of non-overlapping positions at which images of cells pumped into the microfluidic channel will be in the focal plane.
2. Open the "Bioreactor Controller" plugin. Select the desired LED matrix time course, imaging channels, and other experimental settings from the prompts. Collect and analyze images.
3. While the experiment runs, ensure that the media in the media flask remains clear. If it is cloudy, then it has been contaminated.

14. Post-experiment

1. Dispose of media, excess cell culture and effluent.
2. Refill the media flask with 200 mL of 20% EtOH mixed with deionized water. Run the chemostat as it had previously been run during the experiment to wash out cell debris and media.
3. When the alcohol solution has drained from the media flask, disassemble the chemostat fully.
4. Wash glassware and tubes with warm water and mild detergent, and rinse thoroughly with deionized water. Leave to dry.
5. Open the "microcontrollerRecords.csv" file to review the temperature and LED matrix status over the course of the experiment, the "Summary.csv" file to review the summary data from each set of images and the "Results.csv" files to review data summarizing each ROI from each time period, where n is the n^th data set.

Results

This apparatus was used to stimulate a culture of S. cerevisiae expressing yellow fluorescent protein (YFP) in response to blue-light via an inducible optogenetic transcription system based on the CRY2/CIB1 protein pair³⁰. Cells were grown chemostatically in phosphate-limited media with an average dilution rate of 0.2 ± 0.008. Phosphate limitation is commonly used in S. cerevisiae chemostat experiments to control growth rate and the effects of pho...

Discussion

We designed this apparatus with flexibility in mind. All the code used is free and open-source. The default image analysis process to segment cells is simple and runs quickly. Custom analysis could be implemented by recording user input while analyzing a representative image with the FIJI graphic user interface, converting the input to a beanshell script, and then setting the plugin to call the script. When it is called, this script will be sent a String array called "images" containing the file paths to the most...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Extensive lab manual	GitHub	NA	An extensive, regularly updated lab manual is available in the “Optogenetic Chemostat Files” GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files). This also includes a description of the microfluidic mold used to generate the representative results.
Fritzing Design Viewer	Fritzing	NA	The free, open-sourced software to view and edit the .fzz type circuit board designs is available at "http://fritzing.org/download/"
Arduino Uno R3 (Atmega328 - assembled)	Adafruit	50	Microcontroller. 1 required.
Arduino Stackable Header Kit	SparkFun Electronics	10007	Female pin headers for connecting PCB to microcontroller. 1 required.
Adjustable 30W 110V soldering iron - XY-258 110V	Adafruit	180	For making electrical connections to the PCB. 1 required.
Soldering iron stand	Adafruit	150	For making electrical connections to the PCB. 1 required.
Mini Solder spool - 60/40 lead rosin-core solder 0.031" diameter - 100g	Adafruit	145	For making electrical connections to the PCB. 1 required.
0.1 μF capacitor	SparkFun Electronics	COM-08375	Stabilizes voltage in PCB. 1 required.
10 μF capacitor	SparkFun Electronics	COM-00523	Stabilizes voltage in PCB. 1 required.
MAX7219CNG LED Matrix/Digit Display Driver - MAX7219	Maxim	MAX7219CNG	LED driver. 1 required.
8 pin IC Socket	Mouser Electronics	575-144308	16 required. These will be stacked on top of each other to support the culture vessel above the LED matrix.
24 Pin IC socket	Mouser Electronics	535-24-3518-10	Optional. Use this to reversibly attach the MAXIM 7219CNG driver to the PCB.
Digital multimeter	Adafruit	2034	For troubleshooting electronics. 1 required.
Break Away Headers - 40-pin Male (Long Centered, PTH, 0.1")	SparkFun Electronics	PRT-12693	Male pin headers for connected LED matrix to printed circuit board. Ends can be trimmed with wire cutters. 1 set required.
Flush diagonal wire cutters	Adafruit	152	For trimming long pin headers and cutting power cables. 1 required.
Premium Female/Female Jumper Wires - 40 x 12" (300mm)	Adafruit	793	Wire ribbon for connecting breadboard to LED matrix. Can be connected end-to-end with male pin-headers to be longer. 1 required.
Half-size breadboard	Adafruit	64	The LED matrix will connect to this and the culturing vessel will rest above it.
Miniature 8x8 Blue LED Matrix	Adafruit	956	Light source. Dominant wavelength is 470nm (blue). 1 required. Alternative miniature LED matrices from the same vendor are available with dominant wavelengths: 624 nm (red), 588 nm (yellow), 525 nm (green), 572 nm (yellow-green), and white.
Stackable header-3 pin	SparkFun Electronics	13875	8 required.
Resistor Kit - 1/4W (500 total)	SparkFun Electronics	10969	For electronics. 1 required.
IRL520N MOSFET	International Rectifier	IRL520N	Voltage regulating switch for controlling DC current. 4 required.
Hook-Up Wire - Assortment (Solid Core, 22 AWG)	SparkFun Electronics	PRT 11367	Wire for electronics. 1 required.
5V 2A (2000mA) switching power supply - UL Listed	Adafruit	276	Power supply for the heating pad and Arduino. 2 required.
12 VDC 1000mA regulated switching power adapter - UL listed	Adafruit	798	For peristaltic pumps. 2 required.
Electric Heating Pad - 10cm x 5cm	Adafruit	1481	For heating the bioreactor. 1 required.
Low flow variable flow peristaltic pump	Fisher Scientific	13-876-1	For pumping media. 1 required
Medium flow variable flow peristaltic pump	Fisher Scientific	13-876-2	For pumping culture. 1 required.
9 VDC 1000mA regulated switching power adapter - UL listed	Adafruit	63	For microcontroller power supply. Order 1.
High Temp Waterproof DS18B20 Digital temperature sensor + extras	Adafruit	642	Thermometer for the bioreactor. 1 required.
Micromanager	Micromanager	NA	The free, open-sourced microscope control software is available at "https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release"
FIJI	ImageJ	NA	The free, open-sourced image analysis software is available at "http://fiji.sc/"
Arduino Integrated Development Environment	Arduino	NA	The free, open-sourced IDE is available at "https://www.arduino.cc/en/Main/Software"
Custom code	GitHub	NA	The custom microcontroller code and "Bioreactor Controller" plugin are available in the “Optogenetic Chemostat Files” GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).
USB Cable A to B - 6 Foot	SparkFun Electronics	CAB-00512	Used to download data to microcontroller. 1 required.
bioreactorTimecourse_example.csv	GitHub	NA	The advantage of loading LED matrix values from a CSV file is that a program can be called by the plugin to update those values based on image analysis results, and those values can be reloaded to the microcontroller, enabling feed-back control. It is available from the “Optogenetic Chemostat Files” GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).
Tota-frost gels (diffusion paper)	B&H	B&H # LOFSFTL MFR # T1-72	For LED matrix. 1 required.
Kitting Sheet Crosslink 1/4x12x24in	Grainger, inc	20JL37	Black foam for culturing vessel enclosure. 4 required.
Standard Photodiode Power Sensor, Si, 200 - 1100 nm, 50 mW	Thorlabs	S120VC	For measuring light intensity. 1 required.
Labelling Tape	Fisher Scientific	159015N	For labelling and securing loose components. 1 required.
Compact Power and Energy Meter Console, Digital 4" LCD	Thorlabs	PM100D	For measuring light intensity. 1 required.
100mL GL45 hybridization glass bottle	Bellco Glass, Inc.	(7910-40150)	Bioreactor vessel. 1 required.
Six port assembly	Bellco Glass, Inc.	Custom	For the bioreactor vessel. Tubing Specs: .125" OD x .055"ID. Port A: 1.0" long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0" long above cap slug, 33 mm long below. Port D: 1.0" long above cap slug, 65 mm  long below. 1 required. Includes 45 mm diameter polypropylene open top screw cap and a white silicone gasket to ensure a tight seal between the cap and the vessel.
Scotch Magic Tape 3105, 3/4 x 300 Inches, Pack of 3	Amazon	B0009F3P3U	Clear scotch tape. This is available from many other vendors. It is used to cover markings on the culturing vessel and to secure the coverglass with the PDMS channel to the aluminum support frame.
1/16" ID x 3/16" OD x 1/16" Wall Tygon Sanitary Silicone Tubing	United States Plastic Corp.	57288	Tubing. ~25' required.
Cole-Parmer Twistit white rubber stopper, size 10	Cole-Parmer	EW-62992-32	Media flask stopper and effluent flask stopper. 2 required.
2L Laboratory Flask	Pyrex	4980	Media flask and effluent flask. 2 required.
Day pinchcock	Fisher Scientific	5867	For pinching tubes shut. 3 required.
Replacement tubing assembly 1/16" ID	Traceable Products	3372	The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.
Replacement tubing assembly 1/50" ID	Traceable Products	3371	The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.
Male luer with lock ring x 1/16" hose barb, Nylon, 25/pk	Cole-Parmer	EW-45505-00	Connectors. ~10 luers are required.
Male luer with lock ring x 1/8" hose barb, Nylon, 25/pk	Cole-Parmer	EW-45505-04	Connectors. 5 required, one for each rubber stopper hole to fill with tubing.
Female luer x 1/16" hose barb adapter, Nylon, 25/pk	Cole-Parmer	EW-45502-00	Connectors. ~10 luers required.
Female luer x 3/16" hose barb adapter	Cole-Parmer	EW-45502-08	Connectors. ~10 luers required.
Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk	Cole-Parmer	SC-45502-28
Cole-Parmer Luer Accessory, Male Luer Lock Plug, Nylon, 25/Pk	Cole-Parmer	EW-45505-56
Microbore PTFE Tubing, 0.022"ID x 0.042"OD, 100 ft/roll	Cole-Parmer	EW-06417-21	Tubing. 1 roll required.
Masterflex platinum-cured silicone tubing, L/S 13, 25 ft	Cole-Parmer	EW-96410-13	Tubing. ~25' required.
3/16" ID x 1/4" OD x 1/32" Wall Tygon Sanitary Silicone Tubing	United States Plastic Corp.	57293	Tubing. ~1' required.
Vacuum filter	Fisher Scientific	974107	Nalgene vacuum filter for sterile filtering media.
Aquel Oxy-Boost 200	Rena Aquatic Supply	AP200	Dual diaphram adjustable flow air pump for aerating and mixing media. 1 required.
0.2 μm pore syringe filter	Corning International	431229	This ensures that air from the aquarium pump does not contaminate the apparatus. 1 required.
Slygard 184 Silicone Elastomer Kit	Dow Corning	Slygard 184	For microfluidic device. 1 required.
American Safety Razor GEM Scientific Single-Edge Razor Blades	Fisher Scientific	17989000	For cutting tubes and PDMS. 1 blade required.
Harris Uni-Core hole puncher 1.2mm ID	Sigma-Aldrich	WHAWB100028 ALDRICH	For punching inlet/outlet in microfluidic device. 1 required.
Microscope cover glass 22x60-1.5	Fisher Scientific	12-544-G	For microfluidic device. 1 required.
Rectangular aluminum frame with a square window	Custom	Custom	To support the microfluidic channel. Outer dimensions: 3 inches x 1.25 inches. Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches Thickness: ~1/32 inches