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Lab / Wet lab / Experiments

Experiments

An experimental validation of PhytoBlock was carried out to demostrate safety and efficacy. This involved comprehensive testing and molecular characterization of the various parts used for the experiments.

Molecular Biology

Colony PCR using GoTaq® G2 Master Mix from Promega

The aim of this experiment was to validate the occurrence of a specific DNA sequence in bacteria grown on a solid medium. It is commonly used for selecting clones bearing the desired construct after transformation of competent bacterial cells with ligation mixtures. For more detailed guidelines on primer design and technical setup, see [1].
This protocol is based on the guidelines of the manufacturer (Promega), and on a general protocol from Prof. Pinheiro's Lab.

  • GoTaq® G2 Master Mix (catalog nr M7822)
  • Nuclease-free water
  • Primer pair: forward and reverse, 10µM each
    • Equipment
    • Thermocycler
    • Thermoblock
    • Table-top centrifuge

    Procedure

    1. Lysis of bacteria
      1. Fill a 1.5mL tube with 10 µL of nuclease-free water.
      2. Pick the colonies/scrape some material from the plate/tubes with glycerol stocks:
        1. Colonies: After ligation, transformation, and growth of the bacteria, pick 3-8 colonies/variants.
        2. Glycerol stock: Keeping the plate/tube on dry ice, scrape some of the ice and transfer to the PCR tube.
      3. Vigorously wash the tip or toothpick in the PCR tube.
        1. For the colonies: Without contaminating the tip or toothpick, streak it in a LB Agar plate with the corresponding antibiotic.
      4. Lyse the cells for 10 minutes at 95°C, using the thermoblock.
      5. Spin down the debris at 2000-g for 5 minutes.
      6. Use the supernatant as a matrix in your reaction.
    2. Reaction mixture (total volume 25µl)
      Reagent Volume [µl]
      GoTaq® G2 Master Mix 12.5
      10 µM Forward Primer 0.5
      10 µM Reverse Primer 0.5
      Supernatant of the lysed cells 2.5
      Nuclease-free water 9.0

      Note: The reaction volume can be successfully halved to 12.5µl.

    3. PCR reaction steps:
      Temperature Time
      Initial Denaturation 95°C 30 seconds
      25–30 Cycles 95°C 30 seconds
      X 30 seconds
      72°C Y
      Final Extension 72°C 5 minutes
      Hold 16°C forever

      X – annealing temperature; it is recommended to start with temperature 5°C below the lowest melting temperature of the chosen primers.
      Y – elongation time, depends on the size of the expected product and DNA synthesis speed of a chosen polymerase (here, 1 kb/min).

    High-fidelity PCR using Q5® High-Fidelity 2X Master Mix from New England Biolabs

    The aim of this experiment was to amplify a DNA fragment with high fidelity. According to the provider’s website, Q5 polymerase used in this mix offers fidelity over 280x greater than Taq polymerase, significantly lowering the error rate and thus, the introduction of mutations.
    In our project, we used Q5® High-Fidelity polymerase to amplify and add overhangs to our gBlocks. This increased the variety of parts that we could use in the assembly while ensuring an extremely low rate of mutations acquired during amplification.

    Materials

    • GoTaq® G2 Master Mix (catalog nr M7822)
    • Nuclease-free water
    • Primer pair: forward and reverse, 10µM each
    Equipment
    • Thermocycler
    • Thermoblock
    • Table-top centrifuge

    Procedure

    1. General reaction setup
      Component 25 µl Reaction 50 µl Reaction Final Concentration
      Q5 High-Fidelity 2X Master Mix 12.5 µl 25 µl 1X
      10 µM Forward Primer 1.25 µl 2.5 µl 0.5 µM
      10 µM Reverse Primer 1.25 µl 2.5 µl 0.5 µM
      Template DNA Variable* Variable* < 1,000 ng
      Nuclease-Free Water to 25 µl to 50 µl

      In general, we added 5ng of DNA to the 50 µl reaction mixture

    2. PCR reaction steps:
      STEP TEMPERATURE TIME
      Initial Denaturation 98°C 30 seconds
      25–35 Cycles 98°C 5–10 seconds
      X 10–30 seconds
      72°C Y
      Final Extension 72°C 2 minutes
      Hold 4–10°C

    X – annealing temperature of the primers. It is recommended to try the one recommended by the manufacturer (https://tmcalculator.neb.com/#!/main)
    Y – elongation time, depends on the size of the expected product and DNA synthesis speed of a chosen polymerase (here, 1kb/20–30s)

    Restriction Digestion

    Restriction digestion cleaves DNA in sites determined by short, palindromic sequence, recognized by a specific restriction enzyme. This technique is most commonly used in cloning (followed by ligation) or as a method of linearizing a plasmid to verify its size using gel electrophoresis.
    In our project, we used type IIS restriction enzymes in Golden Gate Assembly reactions, or a type II enzyme to verify the size of our assembled plasmids (this protocol).

    Materials

    • rCutSmart™ Buffer (New England Biolabs)
    • Chosen restriction enzyme (New England Biolabs)
    • Nuclease-free water

    Equipment

    • Thermoblock

    Procedure

    1. Prepare the reaction mix. Gently mix the components by pipetting up and down, spin down shortly.
      Component Amount for 25 µl reaction mix [µl] Final concentration/amount
      rCutSmart™ Buffer 2.5 1x
      Restriction enzyme 0.5* Variable*
      DNA sample Variable** Up to 1 ng
      Nuclease-free water Up to 25 µl

      * Enzyme-dependent. Usually, we added 10U of the enzyme of choice to the reaction mixture
      ** Usually, we digested around 500 ng plasmid DNA in the 25 µl reaction

    2. Incubate at the temperature suggested by the provider.
      1. For NEB enzymes: https://www.neb.com/en/tools-and-resources/usage-guidelines/nebbuffer-performance-chart-with-restriction-enzymes?srslitid=AfmBOoryXgkfwwQdjCP_pmRzCALmY04Ce2NNb3c_8u0RcKmZCCoXukLS
      2. Note: In our experiments we used Time-Saver™ Qualified Restriction Enzymes, which allowed us to reduce the restriction digest time to 15 minutes.
    3. Inactivate at the temperature suggested by the provider or add a gel loading dye to immediately run the sample on the gel.

    Golden Gate Assembly – type 0 vectors (BbsI)


    The aim of this protocol was to assemble level 0 vectors with new parts compatible with the SubtiToolKIt. Here, the entry vector (STK001_pSTK-0-sfGFP) and a part of choice (gBlock) are cleaved by BbsI and ligated with T4 ligase.
    The protocol is based on the one from the SubtiToolKit and additional guidelines from our PI. Initially, we noticed a low efficiency of cloning in preparation of our alkaline phosphatase (phoA) plasmids as described in the notebook . After consulting with our PI, we decided to separate the steps of digestion and ligation. To ensure that the ligase is not deactivated during the incubation step, this protocol involves a two-step process whereby the plasmid and insert are digested with the restriction enzyme and then ligated in a second step requiring the addition of the ligase. We tested different incubation time conditions. Notebook .
    Based on the results, we chose 15 minutes incubation with BbsI and 15 minutes incubation with T4 as the incubation times.

    Equipment:

    • Thermoblock
    • Table-top centrifuge

      • Materials

      • BbsI-HF® (R3539S)
      • rCutSmart™ buffer (B6004S)
      • T4 ligase (M0202L)
      • T4 ligase reaction buffer (B0202S)
      • Nuclease-free water

      Procedure

      Restriction
      1. Prepare the reaction mix (total volume 20 µl). To calculate the needed volume of vector and insert, refer to the https://nebiocalculator.neb.com/#!/dsdnaamt calculator.
        Component Volume needed [µl] Final concentration/amount
        Vector variable 50 fmol
        Insert variable 100 fmol
        RCutSmart buffer 10X 2 1X
        BbsI 1
        Nuclease-free water Up to 20 µl
      2. Incubate 15 minutes at 37°C
      Ligation
      1. Prepare the reaction mix with a total volume of 10 µl. Add the ligation mix to the restriction mix.
        Component Volume needed [µl]
        T4 Ligase buffer 10x 3
        T4 Ligase 0.5
        Water 6.5
      2. Incubate for 15 minutes at room temperature
      3. Transform E. coli with the prepared ligation mixture or store temporarily at 4°C

      Golden Gate Assembly – type 1 vectors (BsaI)


      Type IIS enzymes enable effortless assembly of DNA constructs due to cutting a few nucleotides outside of the recognition sequence. Golden Gate Assembly (GGA) is a one-pot, one-reaction cloning method, most frequently involving Type IIS enzymes like BsaI or SapI. The main advantage of GGA is the possibility of designing compatible ends allowing to assemble many DNA parts in the same reaction.
      The SubtiToolKit relies on GGA to clone the parts into full transcriptional units (TUs). Each TU consists of a promoter, ribosome binding site (RBS), coding sequence (CDS; can be split into three parts – Cx, Cy, Cz – to accommodate N- and C-terminal tags) and a terminator. The resulting plasmid is a – shuttle vector between E. coli and Bacillus subtilis. The utilization of a specific syntax enables efficient cloning of the parts in the correct order.
      Described below is the final protocol used by our team to assemble the parts (our troubleshooting steps were further described in the engineering cycle 4 and the notebook ).

      Equipment:

      • Thermoblock
      • Table-top centrifuge

      Procedure

      Restriction
      1. Prepare the reaction mixture
        Component Volume needed [µl] Comments
        DNA parts variable 50 fmol/each plasmid
        RCutSmart buffer 2
        BsaI 1
        Nuclease-free water Up to 10 µl
      2. Incubate for 1 hour in 37°C
      Ligation
      1. Prepare the ligation mixture
        Component Volume needed [µl] Final concentration/amount
        Restriction mixture 10
        T4 ligase (200U/mL) 0.5 50U
        T4 ligase buffer 10X 4 1X
        PEG3000 30% 4 3%
        Nuclease-free water Up to 40 µl
      2. Incubate for 5 minutes in 16°C
      3. Incubate for 20 minutes in 60°C
      4. Transform immediately or store in –20°C

      Chemo competent cells preparation – E. coli DH10b


      This is a standard protocol, adapted by Prof. Pinheiro's lab. The prepared competent cells are stored in –80°C freezer and used in the transformation experiments described below.

      Materials

      • 100 mM CaCl2 solution
      • 100 mM MgCl2 solution
      • 85 mM CaCl2 with 15 % glycerol solution
      • Fresh LB medium without antibiotics

      Equipment

      • Refrigerated centrifuge

      Procedure

      1. Grow overnight culture of desired E.coli strain
        1. Note: we inoculated bacteria from the glycerol stocks in 5 mL LB, cultured in 50 mL falcon tube
      2. Add 1/100 of the overnight culture to fresh LB media
        1. Note: we added 2 mL of the o/n culture to ~200 mL fresh LB
      3. Grow to OD600 = 0.4
      4. Split the culture into falcon tubes, centrifuge at 4°C at 4000 g for 6 minutes
        1. Note: we split our culture into 4 falcons, 50 mL culture each
      5. Discard the supernatant and resuspend pellets in cold 100 mM MgCl2
        1. Note: we resuspended the pellets in 15 mL 100 mM MgCl2/falcon
        2. Avoid pipetting up and down during resuspension – instead, gently tap the falcon on the palm of your hand
      6. Centrifuge at 4°C at 3000 g for 6 minutes
      7. Discard the supernatant and resuspend pellets in cold 100 mM CaCl2
        1. Note: we resuspended the pellets in 2.5 mL 100 mM CaCl2/falcon
        2. Pipet gently up and down
      8. Combine the content of the falcon tubes in one tube
      9. Centrifuge at 4°C at 2000 g for 15 minutes
      10. Discard the supernatant and resuspend the pellets in 2 mL 85 mM CaCl2, 15% glycerol
      11. Aliquot 50 µl/tube and place immediately on dry ice
      12. Store at –80°C

      Transformation– E. coli DH10b


      This protocol is based on the classic protocol for E. coli transformation, adapted by the Pinheiro lab.

      Materials

      • Fresh LB
      • Chemocompetent cells
      • DNA
      Equipment
      • Thermoblock
      • 37°C incubator

      Procedure

      1. Add 5 µl of the cloning reaction mixture to competent cells, on ice
      2. Incubate on ice for 30 minutes
      3. Heat shock at 42°C for 30 seconds
      4. Immediately transfer the cells to ice and incubate for 5 minutes
      5. Add 450 µl of pre-warmed LB medium
      6. Incubate for 60 minutes in 37°C with shaking
        1. Note: in our case, incubation time ranged from 45 to 75 minutes, and the shaking speed was 180 rpm
      7. Plate the cells on the pre-warmed plates
        1. Note: when plating “high” and “low” concentration of cells, we first plated 50 µl of the transformation mixture. Next, we centrifuged the tubes for 3 minutes at 3000 g, discarded the supernatant “in a swing”, resuspended the pellet in the media remaining at the bottom of the tube (around 50 µl) and plated the rest of the mixture
        2. We spread the bacterial cultures on the plates using glass beds. First, we added 5–10 sterile beds onto each plate, then we added the desired volume of the culture onto 1 to 2 beds, closed the lid and shook gently back and forth until the liquid was uniformly spread over the whole plate
      8. Incubate at 37°C

      Chemo competent cells preparation – B. subtilis 168


      Some isolates of B. subtilis can uptake foreign DNA upon reaching stationary growth phase. This state of natural competence is determined by quorum sensing and nutrient limitation [2]. Importantly, not every B. subtilis isolate can be transformed by using the method described below [3]. The protocol below was validated for B. subtilis DB104 [4] and B. subtilis 168 [5]. This protocol is based on the natural competence protocols described by Caro-Astorga et al. [5] and Falkenberg et al [6].

      Materials

      • 10x SM1 medium stock
        • 2 g ammonium sulphate
        • 14 g dipotassium dihydrogen phosphate
        • 6 g potassium dihydrogen phosphate
        • 0.7 g sodium citrate
        • 0.2 g magnesium sulphate heptahydrate
        • 2 g yeast extract
        • 0.25 g casamino acids
        • MiliQ until 100 ml
      • 1x SM2 medium stock
        • 2 g ammonium sulphate
        • 14 g dipotassium dihydrogen phosphate
        • 6 g potassium dihydrogen phosphate
        • 0.7 g sodium citrate
        • 0.8 g magnesium sulphate heptahydrate
        • 1 g yeast extract
        • 0.1 g casamino acids
        • MiliQ until 1 L
      • 200 g/l glucose stock
      • 1M CaCl2 stock
      • 50% glycerol

      Equipment

      • Incubator 37°C
      • -80°C freezer

      Procedure

      1. Grow B. subtilis overnight at 37°C in 1x SM1 medium supplemented with 5 g/L glucose.
        1. First dilute 10x stock with 9 mL sterile H2O. Add 25 µl of the glucose stock per mL of the medium.
        2. Note: we inoculated B. subtilis from a freshly streaked plate into 5 mL LB medium in 50 mL sterile falcon tube.
      2. Measure the OD600 of the overnight culture and dilute it in 250 mL flask to reach OD600 = 0.5 in 10 mL of SM1 medium supplemented with 5 g/L glucose.
        1. Note: for example, if the OD600 of your culture is 1.0, you need to mix 5 mL of your culture with 5 mL of SM1 medium in 250 mL sterile flask.
      3. Grow the culture at 37°C with shaking for 3 hours.
        1. Note: the original protocol mentions 250 rpm as the shaking speed. In our case, we used 180 rpm.
      4. Add 10 mL of SM2 medium followed by 250 µL of 200 g/L glucose stock.
      5. Add 45 µL of the 1M CaCl2 solution while swirling the flask.
      6. Continue incubation at 37°C with shaking for 2 more hours.
      7. Aliquot the cells into 500 µl portions in 2 mL sterile tubes. Add 250 µl of 50% glycerol to the aliquots, mix gently and store at –80°C.

      Transformation – B. subtilis


      This protocol enables the transformation of the competent cells of B. subtilis. It is based on the natural competence protocols described by Caro-Astorga et al. [5] and Falkenberg et al [6].

      Equipment:

      • Incubator 37°C
      • Thermoblock

      Materials

      • LB medium
      • LB agar medium supplemented with desired antibiotic concentration
      • An aliquot of the B. subtilis competent cells
      • Plasmid

      Procedure

      1. Thaw the competent cells on ice.
      2. Add 250 ng–1 pg of plasmid DNA to the competent cells.
      3. Incubate in the thermoblock at 37°C for 30 minutes with 800 rpm shaking.
      4. Add 300 µl of LB medium to each aliquot and incubate at 37°C with 800 rpm shaking for at least 2 hours.
      5. Plate 200 µl of the recovery culture on LB plates
        1. Note: As with E. coli transformation, it is possible to increase the colony count by centrifugation of the samples at 6000g for 2 minutes
      6. Incubate overnight at 37°C.

      Media protocols

      1. V8 Juice Agar (Clarified)


        Purpose

        Plant-juice–based medium for certain fungi/oomycetes.

        Materials

        1. 120 mL V8 juice (unfiltered)
        2. 5 g CaCO3
        3. 12 g Agar
        4. 480 mL tap water

        Protocol


        20% clarified V8-medium
        1. Add 5 g of CaCO3 to 340 mL V8-juice in a beaker containing a stir bar.
        2. Stir for at least 15 minutes.
        3. Aliquot into 50 mL Falcon tubes, making sure the tubes will be balanced in centrifuge – either by weighing or using gradations of tubes.
        4. Spin tubes in a centrifuge at 10,000 rpm for 15 minutes.
        5. Transfer clarified V8-medium to 50 mL aliquots for storage at –20 °C.

        Making of 20% CV8-medium
        1. Add 60 mL of the clarified V8 juice to 240 mL distilled water.
        2. 9 g agar is used for 300 mL liquid.
        3. Mix and autoclave (15 min at 121 °C).

      2. 2. Potato Dextrose Agar (PDA)

        General-purpose fungal medium; typically prepared according to the manufacturer’s label.

        Materials

        1. 39g of Merck PDA powder

        Protocol

        1. Weigh the Merck PDA powder
        2. Add the powder to ~900mL of distilled water in a flask or beaker
        3. Stir and heat gently until fully dissolved, make sure no clumps remain
        4. Bring the total volume to 1L by adding distilled water
        5. Autoclave at 121°C for 15min

      3. 3. Pea Sucrose Agar (PSA)

        Legume extract plus sucrose medium

        Materials

        1. 96g frozen garden peas (no salt!!)
        2. 3g sucrose
        3. 9g agar

        Protocol

        Pea extract
        1. Blend the peas with ~400mL–300mL distilled water until very smooth.
        2. Boil gently for 15–20 min.
        3. Filter through cheesecloth.
        Combine
        1. Combine pea filtrate, 3g sucrose, 9g agar.
        2. Make up the volume to 600mL using distilled water.
        Sterilize
        1. Autoclave 121°C for 15 min.

      4. 4. Carrot Agar (CA)

        Root-vegetable–based medium often used for culturing and sporulation studies of certain plant-associated fungi/oomycetes.

        Materials

        1. 12g potatoes (diced)
        2. 12g carrots (diced)
        3. 9g agar
        4. 600mL distilled water

        Protocol

        Prepare extract
        1. Simmer potatoes and carrots in ~300–400mL distilled water for 30 min
        2. Mash during or after boiling
        3. Strain through cheesecloth to remove solids
        Combine
        1. Add filtrate to a flask, add 9g agar and bring to 600mL volume with distilled water
        Sterilization
        1. Autoclave at 121°C for 15 min

      Assay protocols

      Phytophthora Storage & Revival

      Materials

      • Pipette and pipette tips
      • Round petri plates with media of choice (for example PDA)
      • P. palmivora and P. capsici stock
      • Cryovials with screw caps
      • Cork borer
      • Sterilized tap water (121°C for 20 min)

      Propagation protocol

      1. Transfer a single mycelium plug from the phytophthora stock available, taken with a cork borer, to the centre of round petri plates containing your agar of choice.
      2. Let it grow for 4–10 days.
      3. These plates can now be used for upcoming experiments.

      Long-term storage

      1. If you want to have some long-term storage you can fill a cryovial with 1 mL sterilized tap water and put max. 5 mycelium plugs in the cryovial.
      2. Close the vial with a screw cap.
      3. These can be kept at room temperature (18–25°C) in the shade for max. 6 months.

      Competition assay

      Materials:

      • Round Petri dishes
      • Pipette and pipette tips
      • PSA plates containing Erythromycin and xylose 1%
      • 6-14 days old PDA plates of P. palmivora and P. capsici
      • Liquid bacterial cultures of choice (at least 1 day growth)

      Before starting

      1. Transfer a mycelium plug with a sterile cork borer and forceps from an actively growing mycelium edge of P. palmivora and P. capsici to a new plate of PDA agar. Place the mycelium plug in the centre of the Petri dish. Do this 1 time for each.
      2. Incubate at least 4 days at 27°C.
      3. Start an overnight culture of desired bacteria, 1 day before the start of the experiment.

      Day of experiment

      1. Measure OD of overnight bacterial culture using the Spectrophotometer, desired OD is 0.3
        1. Note: make necessary dilutions if OD is higher the day of the experiments.
      2. Transfer a mycelium plug with a sterile cork borer and forceps from an actively growing mycelium edge of P. palmivora and P. capsici to the centre of a round petri dish containing PSA agar with Erythromycin and 1% xylose.
      3. Then add 20µl (via reverse pipetting) of the bacterial suspension, this is spotted three times, 1.5 cm away from the border of the Petri dish, making sure the angle between each bacterium is ≈120°.
        1. Note: Very important after adding bacterial suspension do not tilt plates! If possible, leave plate to dry 10–15 min before moving out of the hood.
      4. Seal the plates with parafilm.
      5. Incubate 25–27°C for at least 5 days in an incubator.
      6. Analyse plate by taking pictures every 2 hours and tracking growth using ImageJ.

      Protocol: Growth Assay of Phytophthora spp. and B. subtilis on Xylose-Supplemented Agar

      Materials:

      • Phytophthora capsici and Phytophthora palmivora stock cultures (PDA plates)
      • B. subtilis glycerol stock
      • LB Broth
      • Potato Dextrose Agar (PDA)
      • Xylose (3 g per 20 mL water)
      • Distilled water
      • 0.22 µm filter
      • SBS 6-well plates
      • Cork borer (5 mm)
      • Sterile pipette tips
      • Sterile spreader beads

      Preparation:

      Four days prior to experiment:

      1. Plate mycelium plugs of P. capsici and P. palmivora on PDA plates.
      2. Incubate at appropriate temperature to serve as stock cultures for benchmarking growth.

      One day prior to experiment:

      1. Inoculate B. subtilis from glycerol stock into LB broth.
      2. Incubate overnight at 37°C with shaking to reach OD600 ≈ 0.4.

      Agar Preparation

      1. Prepare media according to protocol.
      2. Dissolve 3 g xylose in 20 mL distilled water.
      3. Filter-sterilize xylose solution using a 0.22 µm filter.
      4. Allow PDA to cool to a pourable temperature (~50°C), then add xylose solution and mix gently.
        1. Note: Ensure agar is not too hot before adding xylose to avoid sugar degradation.
      5. Pour media into SBS 6-well plates, filling approximately half of each well.
      6. Allow agar to fully solidify.

      Inoculation

      Phytophthora spp.:

      1. Using a sterile 5 mm cork borer, obtain mycelium plugs from stock cultures.
      2. Place one plug in the centre of four of the agar wells.

      B. subtilis:

      1. Bring overnight culture to OD600 ≈ 0.4
      2. Dilute overnight culture to 10–5.
      3. Spot 50 µL of diluted culture onto sterile spreader beads.
      4. Spread bacteria evenly over the surface of the remaining agar wells.
      5. Incubate the plate at the desired temperature and measure growth by taking pictures every 30 minutes and tracking growth using ImageJ.

      Alkaline phosphatase reporter assay

      Alkaline phosphatase (AP) is a commonly used reporter enzyme in molecular biology. It catalyzes the removal of phosphate groups from various molecules, including nucleotides and proteins. The activity of AP can be easily measured using colorimetric or fluorometric substrates, making it a valuable tool for assessing gene expression and promoter activity in bacterial systems. In this protocol, we describe the steps to perform an alkaline phosphatase reporter assay in B. subtilis.This assay enables the measurement of the activity of alkaline phosphatase (PhoA) produced and secreted by B. subtilis. In brief, bacterial samples are incubated with para-Nitrophenyl phosphate (pNPP). The enzymatic activity is reported by measuring the breakdown of pNPP to yellow para-Nitrophenyl (pNP) (OD410 measurement).

      Equipment:

      • Plate reader
      • Centrifuge
      • 37°C incubator

      Materials:

      • LB
      • 10% xylose solution (filter sterilized)
      • Lysis solution
        • SDS 0.01% (prepare 10% stock solution)
        • 1 mg/ml hen egg lysozyme (store in fridge)
      • Substrate buffer (store in the fridge)
        • 2 mL of 50 mM Tris-HCl, pH=8
        • 2.5 mL of 0.5% pNPP
      • 96-well microplates
        • We used F-bottom Greiner microplates

      Procedure:

      Preparation of cell lysate and culture supernatant samples

      1. Prepare overnight cultures of the tested B. subtilis strains.
      2. In the morning, dilute the cultures in fresh LB medium to OD600 = 0.1
        1. In the cuvette, mix 250 µl of culture and 750 µl of LB
        2. To prevent the contamination of the lab with B. subtilis spores, seal the cuvettes with parafilm since they contain B. subtilis before removing them from the hood
        3. Total volume of the culture should be 5 mL. Prepare 3 replicates/construct
      3. Incubate in 37°C on the shaker until the cultures reach OD600 = 1 (approximately)
        1. Check the OD600 after 1:30h. Afterwards, if needed, check again in half hour increments
      4. Add xylose to 1% final concentration
        1. From a 10% stock of xylose, add 500μl to 5mL culture
      5. Incubate at 37C on shaker for 3 hours.
      6. Centrifuge at 5000g for 10 minutes at room temperature
        1. It is also possible to transfer 1 mL of the culture sample to eppendorf tubes and spin them in a tabletop centrifuge for 5 minutes at 8000g
        2. Keep both supernatant and pellet samples. Transfer supernatant to a new falcon and set aside.
      7. Resuspend pellets in 5mL of Lysis solution
      8. Centrifuge the lysed pellet samples for 15 minutes at 5000g at room temperature; keep the supernatant

      Preparation of the reaction mixture and measurements

      1. To each well in 96-well plate add:
        1. 100uL of the Substrate Buffer
        2. 100uL of the bacterial sample/standard
      2. Use a plate reader device to measure the PhoA activity over time
        1. Our settings: OD410 measurement every 30s for 30 min; incubation in room temperature with shaking
      3. Data analysis

      We analysed our results by preparing two types of graphs:

      • Tracking the accumulation of the coloured product (OD410) over time
      • End-point OD410 measurements, allowing to compare the enzymatic activity in supernatant and cell lysate samples

      Calibration of OD₆₀₀ to CFU for B. subtilis 168

      To establish a correlation between optical density (OD₆₀₀) readings and viable cell counts (CFU/mL) for B. subtilis 168, enabling accurate estimation of bacterial concentration from OD measurements in future experiments.

      Materials

      • B. subtilis 168 overnight culture (grown in LB broth)
      • LB broth (sterile, autoclaved)
      • 500 mL Erlenmeyer flask with grooves (for aeration)
      • Sterile 1.5 mL microcentrifuge tubes
      • Sterile cuvettes
      • Spectrophotometer for OD600 measurement
      • Pipettes and sterile tips
      • Sterile 15 mL conical tubes
      • Agar plates (LB agar)
      • Dilution buffer or sterile LB for dilutions
      • Sterile spreaders

      Procedure:

      Overnight Culture Preparation

      1. Inoculate B. subtilis 168 into 5 mL of LB broth.
      2. Incubate overnight at 37°C with shaking (200 rpm).
      3. Estimating Initial Culture Concentration
      4. Dilute the overnight culture 1:10 in LB broth.
        1. Example: 100 µL culture + 900 µL LB.
      5. Measure OD600 using the spectrophotometer.
      6. Recorded OD600 (1:10 dilution): 0.3
      7. Calculate estimated undiluted OD600:
      8. OD600(original) = 0.3 × 10 = 3.0

      Preparation of Large Culture

      1. Add 50 mL of sterile LB broth to the autoclaved 500 mL flask.
      2. Inoculate with 1 mL of overnight culture.
      3. Incubate at 37°C with shaking.

      Measuring OD600

      1. Obtain a clean cuvette.
      2. Add 900 µL LB broth → place in spectrophotometer → align arrow on cuvette with arrow on machine → press left button to zero (0.000).
      3. Discard LB.
      4. Add 900 µL of culture (or appropriate dilution) to the cuvette.
      5. Insert in same orientation and press right button to record OD600.
      6. Record OD600 at regular time intervals.

      References