TITLE: Use of EcoFAB 2.0 for reproducible plant-microbe interaction experiments
AUTHORS: Vlastimil Novak, Eoghan King, Peter Andeer, John Vogel, Trent t Northen

[DESCRIPTION]
We conducted a multi-laboratory experiment across five labs to demonstrate reproducible microbiome formation, metabolite production, and plant growth using the EcoFAB 2.0 system. This protocol provided standardized instructions for the participating labs to implement the EcoFAB 2.0 system effectively, ensuring consistency in plant-microbiome experimentation. It includes annotated videos detailing each experimental step, utilizing a model bacterial community and the model grass Brachypodium distachyon in a hydroponic EcoFAB 2.0 setup.

[STEPS]
SECTION: Materials and equipment
1. Materials sent by the organizing lab (LBNL)
Material needed for 40 EcoFAB (See EcoFAB 2.0 assembly protocol)
Sterilization pouches
Murashige & Skoog (MS) Basal Salts (Caisson Labs MSP01-1LT).
Micropore tape (UV-treated)
15 mL centrifuge tubes, sterile
2 mL microcentrifuge tubes containing 2 stainless steel beads, sterile
~80 Brachypodium distachyon Bd21-3 seeds
17-member SynCom inoculation solution in 20% glycerol, stored at -80˚C
16-member SynCom inoculation solution in 20% glycerol, stored at -80˚C
Mock solution (½ MS + 20% Glycerol)
Sterile plates, Petri Dishes, 100x15 mm, BD Falcon (sleeve of 25)
3g Phytoagar (Bioworld, Plant Media, #40100072)
2x Box of 50 Acrodisc Syringe Filters with Supor Membrane, Sterile - 0.2 µm, 32 mm (Pall corporation, #4652)
25 kraft paper envelopes 2¼ x 3½ inch
HOBO Pendant Temperature/Light 64K data logger (#UA-002-64)
HOBO Pendant MX Temperature/Light Bluetooth Data Logger (#MX2202)
2 x Vacuum Filter System 1000 mL, 0.22 µm PES Membrane, Sterile (Corning, #431098) – note a vacuum line will be needed for the filtration

 


Additional materials needed
Bleach solution diluted to 6% w/v NaOCl (preferably, to avoid additives in bleach, we use sodium hypochlorite solution VWR, #BDH7038-4L)
70% Ethanol
Sterile Milli-Q water
Sterile 10 mL serological pipettes and matching pipettor
Sterile 1 mL filter tips and matching 1 mL pipette
Sterile 50 mL tubes
Sterile 15 mL tubes
Sterile 1.5 mL tubes
Sterile needles gauge ≥18, length ≥25mm (e.g., DB PrecisionGlide needles #305175)
Sterile 10 mL syringes
Kimwipes
48x LB agar plates with no antibiotics
Medium and fine-tip forceps
Scissors
Timer
Ruler
Glassware (e.g., 1L beaker and bottle) for making plant growth media and storing milli-Q water – make sure all glassware is well washed and baked at 400˚C for 60 min to remove any carbon contamination. Remember to remove tape/marker/pour rings before baking. Alternatively, rinse glassware 3x with Methanol (LC-MS grade, ≥99.9%) 10-20 % of container volume and let it fully dry.

Equipment to be used
Laminar flow hood
Biosafety cabinet (BSC)
Flatbed scanner and Camera
Freezer (-80˚C) and Lyophilizer
Plant growth chamber that is set to 14h light at 26°C / 10h dark at 20°C and Photosynthetic photon flux density (PPFD) to 110-140 µmoles/s/m2 at the plant level. If you can control relative humidity, please set it to 70%. Confirm your growth chamber setting with the PPFD meter and light/temperature sensor.  and insert the values following a link

 

SECTION: Experimental procedure
2. 
 

Timeline (Figure 1)
In advance: Prepare media and supplies, confirm growth chamber parameters
Day -10: Sterilize Brachypodium seeds, and stratify at 4˚C in a fridge
Day -7: Germination of Brachypodium seeds in the growth chamber
Day -4: Setup EcoFABs, image seedlings, and transfer them to EcoFAB
Day 0: Test EcoFAB sterility and inoculate SynCom into EcoFAB.
Day 7: Add sterile Milli-Q water, scan roots
Day 14: Add sterile Milli-Q water, scan roots
Day 22: At the end of the experiment, test EcoFAB sterility, add sterile Milli-Q water, scan roots, sample all growth media, sample and weigh roots, sample and weigh shoots, and store materials.

Layout (Figure 1)
Brachypodium will be grown in EcoFABs (4 treatment groups x 7 replicates = 28):
Axenic plants mock-inoculated
Plants Inoculated with 16-member SynCom
Plants Inoculated with 17-member SynCom
Technical control: ½ MS medium in EcoFAB with no plants, mock-inoculated
Measurements: 
Images of roots and supply of evaporated water are done on days 7, 14, and 22 post-inoculation. 
Sterility tests of uninoculated EcoFABs are done on days 0 and 22.
After 22 days, we will measure root and shoot biomass, collect unfiltered growth media and root
tissues for 16S sequencing, and sample unfiltered media for metabolomics.


 

SECTION: Experimental procedure
3. Prepare in Advance

Assemble and sterilize the EcoFAB 2.0: Follow the EcoFAB 2.0 assembly protocol guidelines and sterilize the devices in individual sterilization pouches (Figure 2, Video 1). 
 

 

 
Video 1: EcoFAB 2.0 assembly

--Work in sterile conditions for the steps below--
Prepare ½ MS media: Dissolve 2.165 g of MS powder (MSP01-1LT) in 1 L Mili-Q water and adjust pH to 5.7 with KOH. Sterile filter the media using the 0.2 µm PES membrane vacuum filtration units. For easy handling, aliquot the media in sterile 50 mL tubes and store it at 4°C.
Prepare 4 plates for seed germination: 1.5% w/v Phytoagar in ½ MS (1.5g/100mL) autoclaved and poured on plates in a sterile hood. Wrap and store plates at 4C°. 
Make 48 LB agar plates for sterility tests: Use 31 plates on Day 0 (28 for all EcoFABs + 3 for neg. control) and 17 plates on Day 22 (14 for control EcoFABs + 3 for neg. control).
Label your EcoFABs and tubes according to the standardized codes X-EF-S, where "X" is the letter assigned to the specific laboratory (lab A-E), "EF" is the EcoFAB number (1-7 for Axenic treatments, 8-14 for SynCom16, 15-21 for SynCom17, and 22-28 for Medium Ctrl.), S is sample type ("Root" for root samples in 2mL tubes, "Med" for filtered medium samples in 15mL tubes, and "16S" for unfiltered medium samples in 1.5mL tubes). 

SECTION: Experimental procedure
4. Day -10: Brachypodium seeds surface sterilization

Dehusk dry seeds: Select seeds that are uniform in size, shape, and color. Remove the lemma with either fingernails or tweezers (Figure 3). The embryo sits at the base of the seed (lemma side), taking care not to damage it. Prepare 40 seeds (germination rate is usually between 80-100% depending on the age of seeds) for the 21 plant-containing EcoFABs
--Work in sterile conditions for the steps below--
Sterilize seeds: Put no more than 25 dehusked seeds into a 2 ml Eppendorf tube. Add 70% ethanol, shake for 30 seconds, and remove the supernatant. Add 1 ml of 6% w/v sodium hypochlorite, shake for 5 minutes, and remove the supernatant. Wash 5 times with 1 mL sterile milli-Q water.
Plate seeds: Using sterilized forceps, place sterile seeds on 1.5% Phytoagar plates (10 seeds per plate) with the embryo (lemma side) facing toward the lid. Wrap the plate with micropore tape and place it in the dark at 4˚C

 

 
Video 2: Brachypodium distachyon seed dehusking, surface sterilization, and plating.


 

SECTION: Experimental procedure
5. Day -7: Brachypodium seeds germination

Transfer plates from the fridge, place them vertically under an angle into a growth chamber (Figure 4), and ensure the embryo poles point down and face the lid. 
 

SECTION: Experimental procedure
6. Day -4: Transfer Brachypodium seedlings to EcoFAB 2.0

Retrieve the germination plates from the growth chamber
Image the plates with seedlings: Take a picture of the plates with a camera (Figure 5)
--Work in sterile conditions for the steps below--
Prepare EcoFABs: Tighten the screws at the back of the EcoFAB and tape the port 1 and air vents according to the assembly protocol. Slowly add 9 mL of ½ MS via port 2 into EcoFAB using a serological pipette. Be careful for the medium not to overflow from the seed port. Seal port 2 with septa.
Transfer the seedlings: Select seedlings that are all uniform in size. Using sterilized forceps, gently grab the seedlings by the seed. Gently insert the root into the plant reservoir port so that most of the root is submerged in the growth medium and the root points towards the opposing sampling port (Figure 5). Take care not to damage the root. If the seedling is damaged, replace it with another. Save the seedling plates to check for contamination at the end of the experiment.
Close the EcoFAB 2.0 and place it with the used seedling plates in a growth chamber.

 

 
Video 3: EcoFAB 2.0 setup and seedling transfer.

SECTION: Experimental procedure
7. Day 0: Add microbes to EcoFABs 2.0

--Work in biosafety cabinet for the steps below--
Check sterility: Temporarily remove the septa. Plate 50 μl of growth medium from each EcoFAB on an LB plate, wrap with a parafilm, and incubate at room temperature. After 7 and 22 days, check for colonies. Be sure to include control plates.
Add Microbes and new growth medium: Thaw SynCom solutions on ice and dilute 100 µL in 9.9 mL sterile ½ MS (in 15 mL tubes). Mix well. Pipet 1 mL of diluted SynCom solution into EcoFAB (Figure 6). If necessary, add sterile Milli-Q water to the refill line (Figure 7). Re-seal the port slowly with the septa.
Add mock solution to plant and medium controls: repeat the process above with the mock solutions.
Tilt to move bubbles: Tilt the EcoFAB slightly to move air bubbles away from the seed port to the septa before returning to the growth chamber.

 

 

 
Video 4: Sterility test and SynCom inoculation into EcoFAB 2.0.

SECTION: Experimental procedure
8. Day 7: Image EcoFABs and refill growth media

Fill with Milli-Q water: In a laminar flow hood, remove the septa and fill EcoFAB with sterile Milli-Q water to the refill line (Figure 7). Reseal port 2 with the septa.
Image the EcoFABs: Scan each EcoFAB to make the root morphology visible. Return the EcoFABs to the growth chamber.
Tilt to move bubbles: Tilt the EcoFAB slightly to move air bubbles away from the seed port to the septa before returning to the growth chamber.
Rotate the EcoFAB position within the growth chamber to minimize the effects of light intensity differences

 
Video 5: Water refill of EcoFAB 2.0.

SECTION: Experimental procedure
9. Day 14: Image EcoFABs and refill growth media

Repeat steps of day 7: Fill with Milli-Q water to the refill line (Figure 7), image the EcoFABs, tilt to move bubbles, rotate the EcoFAB position within the growth chamber
SECTION: Experimental procedure
10. Day 22: Sample EcoFABs — end of the experiment

--Work in biosafety cabinet for the steps below--
Check sterility: Temporarily remove the septa from port 2. Plate 50 μl of growth medium from axenic and technical control EcoFABs on an LB plate, wrap with a parafilm, and incubate at RT. After 7 and 22 days, check for colonies. Be sure to include control plates. 
Sample unfiltered medium for 16S: Pipette 200 μl of growth medium from each EcoFAB into a 1.5 ml tube and store at -80˚C. 
Fill with Milli-Q water: Fill EcoFAB with sterile Milli-Q water to the refill line (Fig. 5). Reseal port 2 with the septa. 
Image the EcoFABs: Scan each EcoFAB so that the root morphology is visible (scanning can be done outside of the BSC). 
Sample growth medium for metabolomics: Uncap the septa. Draw all EcoFAB growth medium with a sterile syringe attached to a needle. Remove the needle, attach a 0.2 µm filter to the syringe, and filter the spent medium into a 15 mL tube (if clotting occurs, replace the filter). Store the filtrate at -80°C.
Harvest shoots and roots from the EcoFAB. Remove the top chamber and discard the seed residue. Gently pull plants slowly up through the seed port, taking care not to damage the plant. Cut the root from the shoot.
Sample roots for 16S. Using sterile tweezers, place roots in pre-weighted 2 mL safe-lock tubes with steel beads, weigh the tube, and subsequently snap-freeze in liquid nitrogen. Sterilize your tolls between each sample to avoid cross-contamination. 
---From here on, there is no need to work sterile anymore. ---
Collect shoots FW phenotypes. Weigh shoots immediately before plants begin to dry. Take pictures of the shoot next to a ruler using a scanner (or a digital camera). Place shoots in individually labeled paper envelopes and freeze them before lyophilizing.
Weight shoot DW out of the envelopes after lyophilization for 3 days.

 
Video 6: EcoFAB 2.0 sampling and harvest

SECTION: Deliverables
11. 
 

Generated data for the LBNL
Weekly root scans in EcoFAB 2.0
Plant biomass table
Pictures of LB agar plates
Pictures of plant shoot phenotypes
Data and pictures should be submitted to the cloud (in this trial, Google Drive) utilizing a common template spreadsheet and file directories to allow for fast sharing of large amounts of data, minimize mistakes, and facilitate downstream analysis. 

Samples shipped to LBNL
Frozen filtered exudates (15 ml tubes on dry ice)
Frozen root tissue (2 ml tubes on dry ice)
Frozen unfiltered media samples (1.5 ml tubes on dry ice)
HOBO data loggers

Downstream processing by LBNL
Root parameter quantification from pictures
Metabolomics on exudates
16S sequencing on roots and medium