Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Caution: This protocol includes the use of hazardous chemicals, sharp objects, electrical devices, hot objects, and other hazards that may result in injury. Appropriate personal protective equipment (PPE, e.g., chemically resistant gloves, safety glasses, lab coat, long clothes, closed-toed shoes, etc.) should be worn, and the appropriate safety procedures (safety training, use of a fume hood, etc.) should be followed.

1. EcoFAB Device Fabrication: Casting PDMS Layers (Figure 2 & Figure 3)

1. Construct the EcoFAB molds using 3D printing techniques (the design files are available at . Each mold includes three parts: a casting frame, a featured mold base, and an insert, as shown in Figure 2. Print the mold base and insert out of rigid opaque photopolymers using a 3D plastic printer. Utilize a minimum of 100 µm resolution, and print the casting frame with acrylonitrile butadiene styrene (ABS).
2. Mix 40 mL of siloxane elastomer base with curing agent in a disposable 1 L container. Depending on the desired experiment (steps 2.1 and 2.2), use different ratios (v/v) of elastomer base/curing agent (5:1, 15:1, or 30:1). Proceed to steps 1.3 to 1.8 for all kinds of mixtures.
   Caution: Wear chemically resistant gloves, safety glasses, and other PPEs.
3. Place the container in a vacuum chamber for at least 30 min to remove air bubbles from the elastomer mixture.
4. Pour the mixture into the assembled 3D printed plastic mold (Figure 3A) and keep the mold on a heating block at 85 °C for 4 h (Figure 3B).
   Caution: Wear PPE to avoid burns.
5. Let the mold cool down for 5 min. Then pull out the insert from the mold gently (Figure 3C), and then slowly insert a utility knife between the casting frame and PDMS (the solidified elastomer mixture) to separate them (Figure 2D).
6. Press the mold base with PDMS up out of the casting frame (Figure 3E). Use a knife or other tools to gently separate the PDMS layer from the mold base at the edges (Figure 3F), and then slowly peel it off from the mold surface (Figure 3G).
7. Create inlet and outlet channels on the PDMS layers by making ~1.6 mm holes for the inlet and outlet ports with a 15 gauge blunt needle (Figure 3H, I).
   NOTE: The standard mold has an inlet and outlet port, while the wide-outlet mold only needs the inlet port (Figure 3H, I).
8. Use scissors to trim the edges of PDMS layers.
   NOTE: The trimmed PDMS layers should be ≥76 mm x 51 mm rectangles for small EcoFAB devices and ≥102 mm x 83 mm rectangles for large EcoFAB devices.

2. EcoFAB Device Fabrication: Chemically Attaching PDMS Layers onto Microscope Slides (Figure 3 & Figure 4)

1. Permanently bonding the PDMS layers to microscope slides
   1. Rinse the bonding side of the PDMS layer (made of a 15:1 elastomer base to curing agent mixture) and 7.6 × 5 cm microscope slide with methanol and then blow dry with compressed air or an ultra-pure nitrogen gun.
      Caution: Methanol is toxic. Work in a fume hood and wear protective eye covering, gloves and other PPEs.
   2. Place the microscope slide and PDMS layer into a plasma cleaner with their bonding sides facing up (Figure 3J). If a plasma cleaner is not available, skip to step 2.2.
   3. Close the chamber and the gas vent valve of the plasma cleaner, and turn on the vacuum and pump down the chamber for 1 min.
   4. Turn on the power of the plasma generator, and switch the radio frequency (RF) level to "HI" for 1 min.
   5. Turn off both the vacuum pump and the plasma power, and vent the chamber to atmosphere.
   6. Take out the microscope slide and PDMS layer from the plasma chamber, and quickly press all four edges of the PDMS layer onto the slide with even pressure (Figure 3L). Make sure the center oval region of the PDMS layer (the root chamber) does not touch the slide.
   7. Place the sealed EcoFAB device on a 120 °C heating block for 20 min to further secure the permanent bonding between the PDMS layer and the microscope slide.
   8. Let the device cool down for 5 min. Trim off the extra edges of the PDMS layer with a knife.
2. Reversible physical sealing of the PDMS layers to microscope slides
   1. The reversible sealing technique utilizes a set of custom clamps (either printed by a 3D plastic printer or machined in metal, the drawings are shown in Figure 4).
      1. Place the microscope slide into the cutout on the bottom clamp plate, and then align the PDMS layer (made of a 5:1 elastomer base to curing agent mixture) on top of the slide.
      2. Place the top clamp plate over the PDMS layer. Secure the top and bottom plates together using four hex cap screws, orienting the screws so that the nuts are threaded on from the top of the clamp.
   2. Adhering PDMS directly to microscope slides
      1. Position the PDMS layer (made of a 30:1 elastomer base to curing agent mixture) on top of a microscope slide.
      2. Press the PDMS layer to the slide. The soft, very adhesive PDMS layer (30:1) should stick to the slide creating a waterproof seal without the permanent chemical bonding or physical press from a clamp (Figure 3L).

3. EcoFABs Sterilization

1. Rinse EcoFAB devices with ultrapure water.
2. Place one EcoFAB device in an EcoFAB container, and add 70% ethanol until the device is submerged. Close the container lid and shake gently to wet all surfaces inside with ethanol. Make sure the root growth chamber of the EcoFAB device is filled with ethanol, with very few or no air bubbles.
3. After 30 min incubation at room temperature, pour off 70% ethanol, and repeat the incubation with 100% ethanol for 5 min.
4. Drain out ethanol, and incubate the sterilized EcoFAB for 16 h in a laminar flow hood to dry it completely. If available, sterilize the system by turning on the UV light within hood for 1 h.
   Caution: Wear appropriate PPE when working with UV lights.
5. Store the sterilized EcoFABs in a sterile hood or autoclaved bags for future use.

4. EcoFABs with LED Grow Lights (Figure 5)

1. Attaching LED strips onto EcoFAB containers
   1. Mark out the locations on the EcoFAB container for 9 LED clips. Start with the first clip 120 mm up from the bottom of the container along an edge (Figure 5A), and proceed to mark out clip locations in a spiral around the container, with each next clip dropping 10 mm. A spiral of 9 clips that allows a 1 m LED strip to wrap around the container twice.
   2. Hot glue an LED clip to each marked position by adding two dabs of hot glue onto the container, aligned with the position of the clips' mounting holes. Press the clip holes into these two dabs of glue, then add another dab of glue on top of the holes. Repeat the procedure for all clips until 9 clips form a spiral (Figure 5B).
      Caution: Wear gloves and other PPE when working with hot glue to avoid burns.
   3. Thread the LED strip through the clips in a spiral shape, with LEDs facing into the container. The strip should circle around twice (Figure 5C).
2. Connecting LED strips to the power supply with a controller (Figure 5D displays an EcoFAB chamber with illuminated lights, the programming of the controller is described in step 4.3).
   Caution: Electrical shock hazard: make sure the power supply is unplugged when connecting wires.
   1. Connect the positive and negative terminals of the power supply to the "INPUT: V+" and "INPUT: V-" terminals of the controller using 2-wire cable (Figure 5E displays a schematic drawing of the controller setup).
   2. Connect the negative lead from the bare end of a female-to-bare cable to one "OUTPUT" channel on the controller.
      NOTE: There are five channels on the controller that is utilized in this protocol, so it can support up to five 1 m LED strips.
   3. Connect all positive leads of the cables to a compact splicing connector (if more than one channels are needed), and then link this connector to the "OUTPUT V+" terminal of the controller.
   4. Plug each LED strip into the female end of the cables, so each LED has its own channel to be controlled. If desired, use female-to-male cables to extend the reach.
3. Program the controller for a desired light cycle according to manufacturer's instructions,

5. Growing Plants in EcoFABs

1. Seed sterilization and germination
   NOTE: The seed sterilization and all following steps with seedlings must be performed in sterile conditions. The sterilization process discussed below is suitable to the seeds of Arabidopsis thaliana, Avena barbata, Brachypodium distachyon, and Panicum virgatum. Panicum virgatum seeds should be suspended in 60% sulfuric acid for 1 h before the sterilization process. It is advised to prepare 1 – 2 seeds per EcoFAB device, considering the germination rate and the homogeneity of germination.
   1. Soak the seeds in 70% ethanol for 2 min.
   2. Remove ethanol with a pipette, and rinse the seeds with sterile water three times.
   3. Leave the seeds in 10% bleach solution for 5 min.
   4. Remove the bleach solution, and thoroughly wash the seeds using sterile water three times.
   5. Add sterile water to the seeds, and incubate the microcentrifuge tube in a 4 °C refrigerator for 7 days.
   6. Evenly spread the seeds on 0.5 Murashige & Skoog (MS) medium with 0.6% phytagel and seal the plates with micropore tape.
   7. Grow the plants to a root length of about 5 mm for transfer to EcoFABs (Figure 6A). For the experiments presented here, apply a 16 h light/8 h dark illumination regime in a 22 °C growth chamber, and incubate the plants 2 – 7 d before transfer to EcoFAB (2 days for Avena barbata and Brachypodium distachyon, 7 days for Arabidopsis thaliana and Panicum virgatum).
2. Transferring seedlings into EcoFABs with liquid medium (Figure 6)
   1. Using a sterile syringe or micropipette, flush the root chamber of an EcoFAB device with sterile water for three times, and then fill the root chamber with the growth medium of interest, for example 0.5 MS medium (Figure 6B, step 5.1.6).
   2. Carefully insert a single seedling into the plant reservoir of the EcoFAB device (Figure 6C).
      NOTE: The root should be fully submerged inside the root chamber, with the shoot sticking out of the reservoir.
   3. Add 3 mL of sterile water into the container, avoiding the EcoFAB device. This will increase the humidity and reduce the evaporation of the medium from the root chamber.
   4. Close the container, and seal the lid with micropore tape (Figure 6D).
   5. Place the EcoFAB into a plant incubator, or utilize the EcoFAB illumination system in a controlled temperature environment suitable for the growth of the respective plant (step 4). For this study, set the chamber to 24 °C.
   6. Periodically check the EcoFAB to refill growth media inside the root growth chamber and add water to the container. Perform all steps in sterile conditions.
      NOTE: For early plant growth stages, refilling of the root growth chamber is necessary every 5 to 7 days. For later growth stages, a refilling is necessary every 2 to 3 days. If desired, use a syringe or pipette to collect root exudate solution from the root growth chambers into an microcentrifuge tube and store it in a -80 °C freezer; also, image the root morphology with a gel imager or microscope.
3. Transferring seedlings into EcoFABs with solid substrates
   1. Use the root chambers fabricated with a 5:1 mixture of elastomer base to curing agent if using a set of custom clamps to attach it to a microscope slide (Figure 3K, Figure 4); or choose a PDMS layer made of 30:1 base to curing agent mixture if adhering PDMS layers to slides directly (as described in step 2.2).
   2. Sterilize the EcoFAB chambers, as described in step 3.
   3. Carefully add sterilized soil/sand into the root chamber, turn the PDMS layer upside down, and add the substrate to the root chamber. Avoid any particulate fall on the area that will be in contact with the microscope slide, since this will reduce adhesion.
   4. Place the microscope slide on top of the PDMS layer, and press all the edges firmly. Carefully flip the EcoFAB device so that no soil/sand falls out of the reservoir opening.
      NOTE: For EcoFAB devices made of a 5:1 base to curing agent mixture, use a custom clamp to secure the seal.
   5. Flow liquid medium or water through the inlet or outlet channel of the EcoFAB device, and transfer a seedling into its plant reservoir, as described in step 3.3.
4. Adding microbes into EcoFABs
   1. Transfer a microbial colony to an incubation tube with 8 mL of LB broth, and grow it to OD 0.5 (approximately 12 h).
   2. Transfer the culture solution into a 15 mL centrifuge tube, and centrifuge it at room temperature for 5 min at 3000 x g to pellet microbes.
   3. Remove the supernatant, and add 8 mL of plant growth medium used in the target EcoFAB. Suspend the pellet of microbes, and centrifuge the tube at room temperature for 5 min at 3000 x g.
   4. Repeat step 5.4.3. twice to completely remove any LB nutrient traces.
   5. Add plant growth medium to the washed microbe pellet until its optical density is about 0.5 at 600 nm.
   6. Add 20 µL of the microbe solution into the root chamber through the EcoFAB outlet. The strains used in this publication traveled to plant roots within 2 – 3 days, and started colonizing root surfaces.
   7. For engineered chemiluminescent, make sure to include the inducer (1 mM IPTG) in plant growth medium to induce luciferase expression.

6. Metabolite Profiling of Root Exudates from EcoFABs

1. Sample preparation for LC/MS based metabolomics analysis
   1. Put the microcentrifuge tubes with root exudates collected from EcoFABs in a lyophilizer, and turn on the lyophilizer to remove all the water from the tubes.
   2. Add 300 µL of LC-MS grade methanol into each tube, and sonicate for 30 min.
      Caution: Wear PPE when working with methanol.
   3. Place the tubes in a centrifuge, and centrifuge them at 3000 x g for 5 min.
   4. Transfer the supernatant solutions into new microcentrifuge tubes, and evaporate methanol in a vacuum concentrator.
   5. Add 150 µL of methanol with 1 mM LC-MS internal standards into each tube, and incubate the tubes in a 4 °C refrigerator for 12 h.
   6. Centrifuge the tubes at 3000 x g for 5 min, and transfer the supernatant into 0.22 µm filter tubes.
   7. Centrifuge the filter tubes, and transfer the filtered solutions into 2.0 mL LC/MS vials with 200 µL of inserts.
   8. Place the vials inside a LC/MS rack, and load the rack inside the LC/MS auto-sampler.
2. Data analysis
   1. Get an access of Metabolite Atlas and custom Python scripts²⁶ or use other data analysis software.
   2. Identify metabolites based on m/z values, retention time and compound fragmentation patterns using a library of metabolite standards.²⁷

7. Mass Spectroscopic Imaging of Plant Roots in EcoFABs (Figure 7)

NOTE: EcoFAB devices made of a 5:1 elastomer base to curing agent mixture with custom clamps (Figure 7A) are used for root stamping onto nanostructure-initiator mass spectrometry (NIMS) chips,^28,29,30 since PDMS layers can be reversely bonded to the surfaces of NIMS chips.

1. Sterilize a NIMS chip surface with UV light for 1 h.
2. Pick an EcoFAB with a growing plant from the incubator, and place it in a sterile hood.
3. Open the EcoFAB container, and remove the top plate of the clamp.
4. Lift up the PDMS layer together with the plant inside, and carefully attach the PDMS layer with the plant onto a NIMS chip (Figure 7B, D, E).
   NOTE: Once the root touches the NIMS chip surface, it should not be moved. This prevents "smearing" of the root metabolites.
5. Gently press down on the roots through the PDMS layer until the roots fully contact the NIMS surface. Leave the roots on the NIMS surface for 20 min.
6. Lift off the PDMS layer including the plant from the NIMS chip, again avoiding moving the root across the NIMS surface. Return the plant to the clamp if desired.
7. Attach the NIMS chip onto a custom MALDI plate and load the plate into a MALDI spectrometer for mass imaging (Figure 7C).
8. Use OpenMSI program to generate the NIMS image of root metabolites (Figure 7D-G)³¹.