Consumables

• Primers (10 µM)
• MilliQ water
• Eppendorf tubes
• PCR tubes
• Template DNA
• Q5 High-Fidelity DNA Polymerase (20.000 unit/mL)
• Q5 HF 2x Master mix

Equipments

• PCR machine

Protocol

Master mix preparation (for all PCR reactions)

Per PCR reaction:

Consumables

• Primers (10 µM)
• MilliQ water
• Eppendorf tubes
• PCR tubes
• Template DNA/Colony of interest
• OneTaq® Quick-Load 2X Master Mix with Standard Buffer dNTPs (10 mM)
• LB agar plate with appropriate antibiotic
• Seeding cove

Equipments

• PCR machine
• MSC

Protocol

Master mix preparation (for all PCR reactions)

Per PCR reaction:

Consumables

• 0.5X TAE buffer
• Agarose (analytical grade)
• Ethidium bromide (stock concentration of 10 mg/mL)

Equipments

• Casting tray
• Well combs
• Voltage source
• Gel box
• UV light source/Gel doc gel photography machine (Bio-Rad Molecular Imager® Gel Doc XR)
• Microwave

Protocol

Making a gel

1. For 0.8% and 1% agarose gel, respectively weigh 2 g and 2.5 g of agarose.
2. Mix agarose powder with 250 mL of 0.5X TAE in a microwavable flask.
3. Microwave until the agarose is fully dissolved. Do not overboil, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. It is best to microwave in pulses and swirl the flask occasionally as the solution heats up.

CAUTION: Hot! Be careful stirring, eruptive boiling can occur.

4. Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask).
5. Pour the agarose into a gel tray with the well comb in place. Pour slowly to avoid bubbles that can disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.
6. Let newly poured gel sit at room temperature until it has completely solidified.

Loading Samples and Running an Agarose Gel

1. Add a loading buffer to each of your DNA samples.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill the gel box with 0.5X TAE until the gel is covered.
4. Carefully load a molecular weight ladder into the first and last lane of the gel.
5. Carefully load your samples into the additional wells of the gel.
6. Run the gel at 100 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 30-40 minutes, depending on the gel concentration and voltage.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8. Stain the gel with Ethidium Bromide on a rocker for 15-30 minutes. If you plan to purify the DNA for later use, cut off the part of interest and store it. Stain the other part with Ethidium Bromide.
9. Use any device that has UV light tovisualize the DNA bands.

CAUTION: Ethidium Bromide is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical.

Consumables

• MACHEREY-NAGEL NucleoSpin® Gel and PCR Clean-up Kit (Binding Buffer NTI, Wash Buffer NT3, Elution Buffer NE, NucleoSpin® Gel and PCR Clean-up Columns, collection tubes)
• MilliQ
• DNA sample to be purified
• 1.5 mL Eppendorf tubes

Equipments

• Centrifuge
• Heating block

Protocol

Sample capture

1. Weigh empty 1.5 mL Eppendorf tube.
2. Cut out the band of interest from gel and place it in an Eppendorf tube.
3. Calculate weight of agarose gel slice.
4. Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g., mix 100 μL PCR reaction and 200 μL Buffer NTI).

Sample binding

1. Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL of sample.
2. Centrifuge for 30 s at 11,000 x g. Discard the flow-through and place the column back into the collection tube.
3. Load the remaining sample if necessary and repeat the centrifugation step.

Wash silica membrane

1. Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g.
2. Discard the flow-through and place the column back into the collection tube.
3. Repeat this step one more time.

Dry silica membrane

• Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.

Elute DNA

1. Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL Eppendorf tube.
2. Add 25 μL Buffer NE and incubate at room temperature (15–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g.
3. Repeat this operation by using the elution volume.

Consumables

• 5X In-Fusion Snap Assembly Master Mix
• Linearized pUC19 control vector (50 ng/μl)
• 2 kb control insert (40 ng/μl)
• MilliQ Water

Equipments

• PCR machine

Protocol

Set up the In-Fusion Snap Assembly cloning reaction into PCR tubes.

Component	Negative control reaction	Positive control reaction	Cloning vector negative control reaction	Cloning reaction
Purified PCR fragment		2 μL of 2 kb control insert		10–200 ng*
Linearized vector	1 μL of pUC19 control vector	1 μL of pUC19 control vector	50–200 ng*	50–200 ng*
5X In-Fusion Snap Assembly Master Mix	2 µL	2 µL	2 µL	2 µL
MilliQ water	to 10 μL	to 10 μL	to 10 μL	to 10 μL

*The optimal amount of insert and linearized vector to use is calculated using the In-Fusion molar ratio calculator.

Incubate the reaction for 15 min at 50°C in the PCR machine, then place on ice. Continue with the E. coli transformation protocol. Cloning reactions can be stored at -20°C until transformation.

Consumables

• Stellar Competent Cells
• 1.5 mL Eppendorf tubes
• Plasmid or In-fusion reaction mixture
• LB agar with appropriate antibiotic plates
• Spreaders

Equipments

• Incubator
• MSC

Protocol

1. Thaw Stellar Competent Cells on ice just before use. After thawing, mix gently to ensure even distribution, and then move 50 μL of competent cells into 1.5 mL Eppendorf tubes. Do not vortex.
2. Add 2.5 μL of the In-Fusion reaction mixture to the competent cells. Place the tubes on ice for 30 minutes.
3. Heat shock the cells for exactly 45 sec at 42°C.
4. Place tubes on ice for 5 minutes.
5. Add 200µL SOC medium. SOC medium should be warmed to 37°C before use.
6. Incubate undershaking (160–225 rpm) for 1 hour at 37°C.
7. Centrifuge samples at 17,000 g for 2 minutes and remove 200 µL of the supernatant.
8. Resuspend the pellet and spread the total volume on a separate LB plate containing an antibiotic appropriate for the cloning vector.
9. Incubate plates overnight at 37°C.

Consumables

• Pseudomonas fluorescens Competent Cells (see competency protocol)
• 1.5 mL Eppendorf tubes
• Plasmid
• LB agar with appropriate antibiotic plates
• Spreaders

Equipments

• Incubator
• MSC

Protocol

1. Thaw P. fluorescens competent Cells on ice just before use. After thawing, mix gently to ensure even distribution, and then move 50 μL of competent cells into 1.5 mL Eppendorf tubes. Do not vortex.
2. Add 2 μL of DNA to the competent cells.
3. Heat shock the cells for exactly 45 sec at 42°C.
4. Incubate on ice for 1 hour.
5. Heat shock the cells for exactly 45 sec at 42°C.
6. Incubate on ice for 5 minutes.
7. Add 200 µL SOC medium. SOC medium should be warmed to 30°C before use.
8. Incubate under shaking (160–225 rpm) for 2 hours at 30°C.
9. Centrifuge samples at 6000 g for 5 minutes and remove 200 µL of the supernatant.
10. Resuspend the pellet in the remaining volume and spread the total volume on a separate LB plate containing an antibiotic appropriate for the cloning vector.
11. Incubate plates for 24 to 48 hours at 30°C.

Consumables

• EcoRI-HF
• HindIII-HF
• Vector/plasmid
• 10X rCutSmart Buffer
• MilliQ water
• PCR tubes

Equipments

• PCR machine

Protocol

For each sample, set up the reactions in PCR tube as following:

Component	Negative control reaction	EcoRI	HindIII	EcoRI + HindIII
DNA sample	1 µg	1 µg	1 µg	1 µg
10X rCutSmart Buffer	2 µL	2 µL	2 µL	2 µL
HindIII-HF (20,000 unit/mL)	0 µL	0 µL	0.5 µL	0.5 µL
EcoRI-HF (20,000 unit/mL)	0 µL	0.5 µL	0 µL	0.5 µL
MilliQ water	to 20 µL	to 20 µL	to 20 µL	to 20 µL

1. Incubate at 37°C for 1 hour.
2. Heat at 80°C for 20 minutes to inactivate the enzymes.
3. Run gel electrophoresis by following the Gel electrophoresis protocol.

Consumables

• DpnI
• PCR product of linearized vector
• 10X rCutSmart Buffer
• MilliQ water
• PCR tubes

Equipments

• PCR machine

Protocol

For each sample, set up the reactions in PCR tube as following:

Component	60 µL reaction
10X rCutSmart Buffer	6 µL
PCR product of linearized vector	50 µL
DpnI (20,000 unit/mL)	1 µL
MilliQ water	3 µL

1. Incubate at 37°C for 1 hour.
2. Heat at 80°C for 20 minutes to inactivate the enzymes.
3. Run gel electrophoresis by following the Gel electrophoresis protocol.

Consumables

• NucleoSpin® Plasmid Mini kit (Resuspension Buffer A1, Lysis Buffer A2, Neutralization Buffer A3, Wash Buffer A4, Elution Buffer AE, RNase A, NucleoSpin® Plasmid Columns, Collection Tubes (2 mL))
• 5 mL overnight liquid culture of transformed E. coli or P. fluorescens
• 1.5 mL microcentrifuge tubes
• MilliQ water

Equipments

• Centrifuge
• Heating block
• Pipettes + pipette tips
• Nanodrop
• Freezer

Protocol

Cultivate and harvest bacterial cells

1. Use 1–5 mL of a saturated E.coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 seconds at 11,000 x g
2. Discard the supernatant and remove as much of the liquid as possible.

Cell lysis

1. Add 250 μL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2! Note: Check Buffer A2 for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30–40 °C until the precipitate is completely dissolved. Mix thoroughly and cool the buffer down to room temperature (18–25 °C).
2. Add 250 μL Buffer A2. Mix gently by inverting the tube 6–8 times. Do not vortex to avoid shearing of genomic DNA. Incubate at room temperature for up to 5 minutes or until the lysate appears clear.
3. Add 300 μL Buffer A3. Mix thoroughly by inverting the tube 6–8 times until blue samples turn colorless completely! Do not vortex to avoid shearing of genomic DNA.

Clarification of lysate

1. Centrifuge for 5 minutes at 11,000 x g at room temperature.
2. Repeat this step in case the supernatant is not clear!

Bind DNA

1. Place a NucleoSpin® Plasmid/Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 700 μL of the supernatant onto the column. Centrifuge for 1 minute at 11,000 x g.
2. Discard the flow-through and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the collection tu

Wash silica membrane

1. Add 600 μL Buffer A4. Centrifuge for 1 minute at 11,000 x g.
2. Discard the flow-through and place the NucleoSpin® Plasmid / Plasmid (NoLid) Column back into the empty collection tube.
3. Repeat this step another time.

Dry silica membrane

• Centrifuge for 2 minutes at 11,000 x g and discard the collection tube.

Elute DNA

1. Preheat Buffer AE at 70°C.
2. Place the NucleoSpin® Plasmid / Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 μL Buffer AE. Incubate for 1 minute at room temperature.
3. Centrifuge for 2 minutes at 11,000 x g.

Consumables

• NucleoSpin® Plasmid Mini kit (Lysis Buffer T1, Lysis Buffer B3, Wash Buffer BW, Wash Buffer B5, Elution Buffer BE, Proteinase K, Proteinase Buffer PB NucleoSpin® Tissue Columns, Collection Tubes (2 mL))
• 5 mL overnight liquid culture of P. fluorescens
• 1.5 mL microcentrifuge tubes
• MilliQ water

Equipments

• Centrifuge
• Heating block
• Pipettes + pipette tips
• Nanodrop
• Freezer

Protocol

Prepare sample

1. Resuspend up to 107 cells in a final volume of 200 μL Buffer T1.
2. Add 25 μL Proteinase K solution and 200 μL Buffer B3. Vortex to mix and incubate the sample at 70°C for 10 to 15 minutes.

Pre-lyse sample

1. Add 180 μL Buffer T1 and 25 μL Proteinase K solution.
2. Vortex to mix. Be sure that the samples are completely covered with lysis solution. Incubate at 56°C until complete lysis is obtained (at least 1 – 3 h). Vortex occasionally during incubation or use a shaking incubator.

Lyse sample

1. Vortex the samples. Add 200 μL Buffer B3, vortex vigorously and incubate at 70°C for 10 minutes.
2. Vortex briefly.

Adjust DNA binding conditions

• Add 210 μL ethanol (96 – 100 %) to the sample and vortex vigorously.

Bind DNA

1. For each sample, place one NucleoSpin® Tissue Column into a Collection Tube. Apply the sample to the column.
2. Centrifuge for 1 minute at 11,000 x g.
3. Discard Collection Tube with flowthrough and place the column in a new Collection Tube.

Wash silica membrane

1^st wash

• Add 500 μL Buffer BW. Centrifuge for 1 minute at 11,000 x g. Discard the flowthrough and place the column back into the Collection Tube.

2^nd wash

• Add 600 μL Buffer B5 to the column and centrifuge for 1 minute at 11,000 x g. Discard the flowthrough and place the column back into the Collection Tube.

Dry silica membrane

• Centrifuge the column for 1 minute at 11,000 x g.

Elute highly pure DNA

1. Place the NucleoSpin® Tissue Column into a 1.5 mL microcentrifuge tube and add 100 μL Buffer BE.
2. Incubate at room temperature for 1 minute.
3. Centrifuge 1 minute at 11,000 x g.
4. Elute highly pure DNA Place the NucleoSpin® Tissue Column into a 1.5 mL microcentrifuge tube and add 100 μL Buffer BE.
5. Incubate at room temperature for 1 minute.
6. Centrifuge 1 minute at 11,000 x g.

Consumables

• Gel SDS-Page precast : Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gels (BioRad)
• Buffer A : 0.1 M Sodium Phosphate, 0.3 M NaCl pH 7.4
• Buffer B: 0.1 M Sodium Phosphate, 0.3 M NaCl, 8 M Urea pH 7.4
• 2 mL lysis matrix B tubes (MP biomedicals)
• 4X Laemmli Buffer supplemented with 50 mM Dithiothreitol (DTT)
• TGS 1X buffer : 2.5 mM Tris, 19.2 mM glycine, 0.01% SDS, pH 8.3 (Biorad)
• Precision Plus protein Unstained blue protein standard (Biorad)

Equipments

• Voltage source
• FastPrep 24 5G

Protocol

Bacteria production & protein extraction

1. Grow a 25 mL culture in LB overnight at 30°C / 140 RPM.
2. Pellet the cells and resuspend in buffer A to a final OD of ~25.
3. Add to a 2 mL lysis matrix B tubes resuspended cells to a final OD of ~ 5.
4. FastPrep the cells for 30 seconds at 6 m/s.
5. Centrifugate at 17 500 g for 15 minutes.

Soluble protein analysis

1. Collect the supernatant and go step “Protein sample preparation”.

Insoluble protein analysis

1. Resuspend the pellet in 1 mL of Buffer B and heat to 37°C for 1h00 under mild agitation
2. Centrifugate at 17 500 g for 15 minutes.
3. Collect the supernatant and go to step “Protein sample preparation”.

Protein sample preparation

• Mix 30 µL of the protein sample with 10 µL.
• Heat 95°C for 10 minutes.

SDS-Page running & revelation

1. Load 15 µL of each sample.
2. Load 8 µL of Precision Plus protein Unstained blue protein standard.
3. Run migration for 30 min at 200V.
4. Reveal the gel with StainFree procedure (2 minutes activation) under the SafeImager (Biorad) or colored using Instant Commassie Blue (15 minutes at room temperature under mild agitation followed by 1h00 decoloration step).

Consumables

• LB medium
• Culture of Pseudomonas fluorescens
• M9 citrate medium
• Sterile distilled water
• Crystal violet
• Paper towels
• Ethanol
• Acetate

Equipments

• Sterile 100 mL Erlenmeyer
• 96-well plate with U-shaped bottom
• 96-well plate with flat bottom
• Tub of water
• Gloves, lab coat
• Biosafety cabinet
• Pipettes, sterile tips
• UV plate reader

Protocol

Biofilm development

1. Grow a culture of the wild-type Pseudomonas fluorescens or mutant strain overnight in a rich medium (i.e., LB)
2. Dilute the overnight culture 1:100 into fresh medium for biofilm assays (here KB medium)
3. Add 100 μL of the diluted culture per well in a 96-well U-shaped plate. For quantitative assays, we typically use 4-8 replicate wells for each treatment.
4. Incubate the microtiter plate for 4-24 hours at 30°C.

Biofilm staining

1. After incubation, dump out cells by turning the plate over and shaking out the liquid.
2. Gently submerge the plate in a small water tub. Shake out water. Repeat this process a second time. This step helps remove unattached cells and media components that can be stained in the next step, and significantly lowers background staining.
3. Add 125 μL of a 0.1% solution of crystal violet in water to each well of the microtiter plate. Wear gloves and a lab coat while making the solution. Use caution when weighing out the crystal violet as the powder is hydroscopic and readily stains clothing, skin, etc.
4. Incubate the microtiter plate at room temperature for 10-15 min.
5. Rinse the plate 3-4 times with water by submerging in a water tub as described above, shake out and blot vigorously on a stack of paper towels to rid the plate of all excess cells and dye.
6. Turn the microtiter plate upside down and dry for 2 to 3 hours.
7. For qualitative assays, the wells can be imaged when dry

Biofilm quantification

1. Add 125 μL of a solution of 80% ethanol and 20% acetone to each well of the microtiter plate to solubilize the crystal violet.
2. Incubate the microtiter plate at room temperature for 10-15 min.
3. Transfer 125 μL of the solubilized crystal violet to a new flat-bottom microtiter plate.
4. Quantify absorbance in a plate reader at 540 nm using a solution of 80% and ethanol 20% acetone as the blank.

Consumables

• Autoclaved regolith simulant
• Potting soil
• Sterile 1.5 mL Eppendorf tubes
• Distilled water
• 0.7 mm needle
• 15 mL Falcon tubes
• 1µm pore diameter filters
• Biofilm forming bacteria
• Biofilm non forming bacteria
• M9 minimal medium supplemented with glucose (28 mM)

Equipments

• Centrifuge with 15 mL falcon adaptor
• Gloves, lab coat
• Biosafety cabinet
• Pipettes, sterile tips
• Gloves, lab coat

Protocol

Preculture

1. Inoculate Erlenmeyer flasks containing 20 mL of M9 minimal medium supplemented with glucose (28 mM) with biofilm forming and non forming bacteria.
2. Incubate the flasks overnight at the optimal growth temperature, shaking at 150 RPM.
3. Measure the optical density at 600 nm (OD600 nm) of the preculture.
4. Inoculate 1 mL of M9 minimal medium supplemented with glucose (28 mM) with an appropriate volume of the preculture to achieve a final OD600 nm of 0.2. Also inoculate a tube with only M9 minimal medium supplemented with glucose (28 mM) for the negative control of the experiment.

Growth phase

1. Place a filter at the bottom of each pierced Eppendorf tube.
2. Weigh 0.5 g of regolith and transfer it into Eppendorf tubes.
3. Inoculate the Eppendorf tubes containing regolith with 1 mL of the 0.2 OD600 nm solution. Seal the tubes with cotton to allow air exchange and incubate at the optimal temperature until the water has completely evaporated.
4. Pierce the bottom of each Eppendorf tube with a needle.

Measurement

1. Add 800 µL of distilled water to each Eppendorf tube.
2. Weigh the same number of Falcon tubes as there are Eppendorf tubes and place the closed Eppendorf tubes inside them.
3. Centrifuge the Falcon tubes containing the Eppendorf tubes at 5 g for 5 minutes.
4. Remove the Eppendorf tubes and reweigh the Falcon tubes.
5. Calculate the mass difference to determine the amount of water retained by the soil or regolith.

E. coli K-12 W3110 WT cultures were grown at 30°C under 150 rpm orbital shaking, in 100mL flasks containing 20 mL M9 without nitrogen source. Samples (500 μL) were collected every 60 min. Bacteria were immediately removed by centrifugation (and the flow-through was kept at −20°C until further analysis.

The flow-through was thawed and mixed (180 μL) with 20 μL of an internal standard containing 2.35 g/L deuterated trimethylsilylpropanoic acid (TSP-d4) solubilized in D2O. The 1H-NMR analyses were performed using a quantitative zgpr30 sequence with water pre-saturation prior to acquisition on an Avance III 500-MHz spectrometer. The parameters were as follows: 286K, 128K points, 4 dummy scans, 32 scans, interscan delay of 8.98 s. For the ammonium quantification experiment, the filtration flow through (250 μL, was mixed with 2.4 μL 4 M H2SO4 and 1.2 μL MQ H2O. TSP-d4 dissolved in D2O (20 μL) was added to 180 μL of the mixture. A solvent suppression proton NMR sequence (zggpw5) at 286K with 32K points, 32 scans, 4 dummy scans, and an interscan delay of 10 s was then applied. Quantification was achieved through an external NH4Cl standard curve (50 mM, 20 mM, 10 mM, 5 mM, and 2mM NH4Cl diluted in MQ H2O) analyzed with 1H-NMR with the same acquisition parameters and the same sample preparation. Quantification of ammonium in solution was achieved by integration of NMR signals of protons (between 6.9 and 7.4 ppm) attached to nitrogen in an acidic medium, according to the different occurring species (i.e., NH4, NH3D, NH2D2) in a mixture of H2O and D2O (NHD3 and ND4 in negligible amounts were not detected).

Spectrum analysis was performed using topspin version, and further data were interpreted using physiofit.

Consumables

• LB medium
• M9 minimal medium with glucose (28 mM)
• Sterile osmotic water
• Streptomycin
• m-Toluic acid
• Bacterial glycerol stock
• Physiological water
• 50 mL Falcon tubes
• Spectrophotometer cuvette
• Pipettes
• 100 mL and 250 mL erlenmeyers
• Inoculation loop

Equipments

• Spectrophotometer
• Centrifuge
• 96-well plate with flat bottom
• Etuve
• Biosafety cabinet

Protocol

1. Inoculate from Pseudomonas fluorescens glycerol stock 100 mL culture erlenmeyers containing 20 mL of LB medium and antibiotic, when necessary.
2. Incubate overnight at 30°C under agitation at 150 RPM.
3. Measure the optical density at 600 nm (OD600 nm) of the preculture (PC).
4. Inoculate 50 mL of M9 minimal medium supplemented with glucose (28 mM) and necessary antibiotic to achieve an initial OD (ODi) of 0.1 UA, following the calculus:

V_PC =

V _{M9 culture}*OD_initial / OD_PC
5. Incubate at 30°C under shaking at 150 RPM and monitor the OD and the growth of this culture by taking 750 µL each hour.
6. Stop the culture when it achieves the end of the exponential phase (OD=1.2 UA).

For a 6-wells regolith plate:

1. Centrifuge 6 mL of the culture at 4,000 RCF for 10 minutes at 25°C in a 50 mL falcon tube.
2. Discard the supernatant.
3. Wash the cells by resuspending them with 6 mL of physiological water, centrifuge at 4,000 RPM for 10 minutes at 25°C.
4. Discard the supernatant.
5. Resuspend the pellets in 1,2 mL of osmotic water. This is the inoculate solution.
6. Inoculate each well with 200 µL of the inoculate solution at the bottom of the stem, taking care not to touch the leaves, this will roughly translate to 108 cells per plant.

Consumables

• Dry autoclaved regolith
• Sterile soil
• Creatinine solution (44 mM) made with osmotic water
• Omni tray single-well plate
• Parafilm
• 24 wells plates
• Osmotic water
• Sterile water
• Bleach 5%
• Ethanol 70%
• 5mL Eppendorf tube
• Pipettes
• Tweezers
• Protective mask

Equipments

• Lighting system composed of GC 4 LED-Module from Greenception used in mode full spectrum (350 - 780 nm)
• Biosafety cabinet
• Drill

Protocol

Germination:

1. Under sterile conditions in a 5 mL Eppendorf tube filled with ethanol, put approximately 200 seeds for 1 mL of ethanol 70%. Shake several times.
2. Remove ethanol and put 1 mL of 5% bleach solution for 5 minutes. Remove the bleach solution.
3. Wash the seeds by adding 1 mL of sterile water and remove the water. Repeat this step 5 times. Keep the water at the final step.
4. Spread directly the seeds solution with a pipette on a square Petri dish filled with 5 mm of sterile soil.
5. Water it with 5 mL of osmotic water.
6. Put the lead on and maintain it with Parafilm.
7. Place the Petri dish under the lighting system with a photoperiod setting to 10/14 hours of day/night for at least one week.

Preparation of the plate:

1. Using a small drill, drill four holes, each 3mm in diameter, under each well of a 6-well plate.
2. Place a 0,2 µm filter at the bottom of each well.
3. Fill each well of a 6-wells plate with 14 g of filtered and sterile regolith.
4. For the positive control, fill each well of a 6-wells plate with 12 g of sterile soil.

Planting:

1. When the seedlings have the two first leaves (after the two cotyledons), remove them gently from the soil with tweezers to keep the root system unspoiled.
2. Wash the roots with osmotic water to remove soil residues.
3. Dig a small hole on the regolith/soil.
4. Plant the seedlings taking care to cover the roots well with the regolith/soil and water immediately with 3 mL of osmotic water.
5. Inoculate well with 200 µL of P. fluorescens inoculated watering solution (prepared following “Preparation of the Pseudomonas fluorescens inoculated watering solution” protocol) or osmotic water.
6. Because seedlings are transferred from a Petri dish with 100% humidity to the ambient air, put a lid above the planted microplates to keep humidity to avoid extra stress. Remove slowly this lid until day three when it can be completely removed.
7. Set the photoperiod to 10/14 hours of day/night.
8. Maintain the humidity between 55% and 60% and check it with a hygrometer.
9. Water at the base of the stem every 24 hours during the night period with 3 mL of osmotic water.
10. Every three days, water the plants with 3 mL of the creatinine solution (44 mM) previously tree time diluted.
11. Photograph the plants every day and count the number of leaves as well as the number of wilted leaves.

From these photos, rosette area and color were analyzed daily using ImageJ.

Consumables

• LB medium
• M9 minimal medium with glucose (28 mM)
• Sterile osmotic water
• Streptomycin
• m-Toluic acid
• Bacterial glycerol stock
• Physiological water
• 50 mL Falcon tubes
• Spectrophotometer cuvette
• Pipettes
• 100 mL and 250 mL erlenmeyers
• Inoculation loop

Equipments

• Spectrophotometer
• Centrifuge
• 96-well plate with flat bottom
• Etuve
• Biosafety cabinet

Protocol

1. Inoculate from Pseudomonas fluorescens glycerol stock 100 mL culture erlenmeyers containing 20 mL of LB medium and antibiotic, when necessary.
2. Incubate overnight at 30°C under agitation at 150 RPM.
3. Measure the optical density at 600 nm (OD600 nm) of the preculture (PC).
4. Inoculate 50 mL of M9 minimal medium supplemented with glucose (28 mM) and necessary antibiotic to achieve an initial OD (ODi) of 0.1 UA, following the calculus:

V_PC =

V _{M9 culture}*OD_initial / OD_PC
5. Incubate at 30°C under shaking at 150 RPM and monitor the OD and the growth of this culture by taking 750 µL each hour.
6. Stop the culture when it achieves the end of the exponential phase (OD=1.2 UA).

For a 6-wells regolith plate:

1. Centrifuge 6 mL of the culture at 4,000 RCF for 10 minutes at 25°C in a 50 mL falcon tube.
2. Discard the supernatant.
3. Wash the cells by resuspending them with 6 mL of physiological water, centrifuge at 4,000 RPM for 10 minutes at 25°C.
4. Discard the supernatant.
5. Resuspend the pellets in 1,2 mL of osmotic water. This is the inoculate solution.
6. Inoculate each well with 200 µL of the inoculate solution at the bottom of the stem, taking care not to touch the leaves, this will roughly translate to 108 cells per plant.