Team:MichiganState/Experiments

Experiments

Protocols:

Endophyte isolation from switchgrass

Materials

  • Small shovel
  • Small baggies
  • Metal tray
  • Aluminum foil
  • Tweezers
  • Scissors
  • TWEEN 20
  • ddH2O
  • Sterile filter paper
  • King B agar
  • 70% ethanol
  • 50% bleach
  • Sterile coffee filters
  • Sterile 0.85% saline solution

Methods

  1. Plant collection
    1. Extract plants along with their roots from soil by digging around roots with a small shovel and pulling up
    2. Store plants in small baggies overnight at 4°C
  2. Root cleaning
    1. Clean roots on the platform of a metal tray covered with aluminum foil that can be switched out between plants
    2. Remove plants from the baggies with ethanol sterilized tweezers and place on the tray, gently shaking off as much soil from the roots as possible
    3. Cut off the thinner roots with ethanol sterilized scissors to save because these roots are more metabolically active
    4. Place thin roots in 50 ml conical vials for subsequent washes. All of the following washes were done by filling up the vials and both inverting a vortexing:
      1. 3 TWEEN 20 washes
      2. 3 ddH2O washes
    5. Dry off the clean roots with sterile filter paper
  3. Root sterilization
    1. Controls
      1. Positive: Plate a single root on King B agar before sterilization and incubate at 30°C for 48 hours
      2. Negative: Plate a single root on King B agar after sterilization and incubate at 30°C for 48 hours
    2. All of the following washes were done by filling up the vials and gently inverting for 1 min each:
      1. 1 70% ethanol wash
      2. 1 50% bleach wash
      3. 5 ddH2O washes
    3. Transfer sterilized roots sterile coffee filters and pat dry
    4. Cut up roots into 1 cm segments using flame sterilized scissors
    5. Incubated these root segments in 0.85% saline solution at 30°C for 48 hours
  4. Endophyte plating
    1. 12 sterile culture tubes with 9 ml of 0.85% saline (series A-C) one set for Fertilized one set for unfertilized
      1. 3 tubes of 10-1 : 0.5 ml of 100 suspension + 5 ml of 0.85% solution
      2. 3 tubes of 10-2 : 0.5 ml of 10-1 suspension + 5 ml of 0.85% solution
      3. 3 tubes of 10-3 : 0.5 ml of 10-2 suspension + 5 ml of 0.85% solution
      4. 3 tubes of 10-4: 0.5 ml of 10-3 suspension + 5 ml of 0.85% solution
    2. Spread plates (100μl of culture used)
      1. 100
      2. 3 series (A-C) each have the following dilutions: 10-1, 10-2, 10-3, 10-4
    3. Streak plates
      1. 100
      2. 3 series (A-C) each have the following dilution: 10-1
    4. Incubate plates at 30°C overnight

Fast-extraction of fungal tissue or bacterial colonies

Materials

  1. Extraction solution (ES)
    • 5 mL of 1 M Tris (pH 9)
    • 0.93 g KCl
    • 0.19 g Na2-EDTA
    • 50 mL dH2O
  2. Titrate with 1 M NaOH to pH ~ 9.5-10
  3. Filter sterilize into 2 mL microcentrifuge tubes
  4. 3% m/v bovine serum albumin (BSA), filter sterilized
  5. PCR tubes
  6. P200 tips
  7. Fungal or bacterial culture growing on solid media

Methods

  1. Add 20 μl of ES to PCR tubes.
  2. For fungi
    1. Heat-sterilize forceps and allow to cool.
    2. Use forceps to remove a very small tuft of mycelium from the fungal culture, place into PCR tube with ES. Swirl to remove it from forceps.
  3. For bacteria
    1. Use a 200 μl pipet tip to lightly touch a colony.
    2. Place tip into the PCR tube with ES. Swirl for 10 seconds.
    3. Close the PCR tube, and place in thermocycler for 10 minutes at 95°C.
  4. Add 40 μl BSA to each sample. This solution is now good template for PCRs.
  5. Store at -20°C.

Brachypodium seed sterilization and germination

Materials

  • 0.6% active bleach(10% bleach to distilled water), 0.1% Tween 20, 50 mL
  • Brachypodium distachyon seeds, looked over to ensure that seeds are fertile
  • Sterile distilled water
  • Strainer
  • 50 mL conical centrifuge tube
  • MS/2 agar plates (Murashige and Skoog 1962)
  • Forceps
  • Aluminum foil

Methods

  1. Make bleach/Tween 20 solution in 50 mL tube.
  2. Add seeds to solution, time for 7 minutes while inverting.
  3. Pour out seeds against strainer, put back into tube.
  4. Add about 30-40 mL of sterile distilled water, shake and pour out.
  5. Repeat step 4 for a total of 5 rinses, place seeds back into tube.
  6. Heat sterilize forceps, allow to cool.
  7. Use forceps to place seeds into the agar plates, submerging then below the surface.
  8. Seal plates, then cover with aluminum foil.
  9. Put plates at 4°C for 72 hours.
  10. Remove plates, put at about 22°C for 48 hours.
  11. Transplant germinated seeds to choice container.

Making electrocompetent Enterobacter ludwigii FCP2-01

Materials

  • Sterile distilled water
  • 50 mL conical centrifuge tubes
  • 100 mL overnight culture of FCP2-01 in LB, shaking at 225 rpm at 30°C for 15-24 hrs, OD600 ~6-7
  • 10% glycerol, sterile
  • Sterile microcentrifuge tubes, flat bottom may be desirable to improve mixing of competent cells

Methods

  1. Pour or pipet 50 mL of overnight culture into each centrifuge tube.
  2. Centrifuge at 4000 x g for 10 minutes.
  3. Pour off supernatant, add 25 mL of distilled water and vortex to resuspend.
  4. Centrifuge at 4000 x g for 10 minutes.
  5. Pour off supernatant, add 25 mL of 10% glycerol.
  6. Aliquot to either use immediately, or into microcentrifuge tubes.
  7. Freeze cells at -80°C to use later. Thaw on ice to use.

Transformation of Enterobacter ludwigii FCP2-01

Materials

  • Electrocompetent FCP2-01 cells
  • 1 mm electroporation cuvettes, chilled on ice
  • Plasmid DNA, works well with 100 ng per sample
  • SOC media
  • LB plates with antibiotic for selection

Methods

  1. Thaw FCP2-01 cells on ice if using them out of the freezer.
  2. Add 100 μl of cells to cuvettes on ice.
  3. Add ~ 100 ng of plasmid DNA to cells, incubate on ice for 30 minutes.
  4. Electroporate at 1.8 kV, add 1 mL SOC.
  5. Incubate for 1 hr in cuvette at 30°C, warm plates to 30°C.
  6. Spread 100 μl of cells on LB plates, incubate 24-48 hrs to see colonies.

Note: Some colonies grow which may not be transformed. It is therefore useful to include a fluorescent protein marker.

Characterization of endophytic isolates

Optical Density (OD) at 600nm as measure of growth rate

  • Overnight cultures in 6 mL LB were inoculated from colonies on solid media and grown for 24 hours at 30°C. These cultures were then diluted to a standard OD and a plastic inoculation loop was used to inoculate a second 6 mL LB culture. After 24 hours at 30°C, The 1/10 diluted OD was measured.
Susceptibility
  • Cultures grown overnight at 30°C in 6 mL LB were streaked to form a lawn on MH plates. Sterile paper disks were soaked in either 35 mg/ml chloramphenicol or came pre-treated with streptomycin. The disks were placed on the streaked plates are incubated at 30°C for 24 hours. The radius of clearance was measured are compared to standards.

ACC utilization

  • Overnight cultures of isolates were streaked onto DF-ACC agarwithout MoO3 to prevent nitrogen acquisition via fixation. After 2 days on these plates, presence or absence of growth was recorded.

Sequencing

  • The 16S region of the bacterial isolates was amplified using the universal bacterial primers 27F and 1492R. Polymerases Q5, Phusion, or Taq were used for amplification. PCR products were processed for sequencing via reaction with Exonuclease I and Antarctic Phosphatase. Sanger sequencing using 27F was performed at the MSU Genomics Core.
Endophyte persistence assay Materials
  • 15 ml conical vial
  • Luria Bertaini (LB) broth
  • Sterile 0.85% saline solution
  • Sterile water
  • Sterile 38 x 200mm culture tubes
  • Sterilized potting mix
  • Brachypodium distachyon seeds
  • TWEEN 20

Methods

  1. Cell Culture
    1. 200 ml baffled flasks with 25 ml of LB broth, 35 ug/ml chloramphenicol
    2. Inoculate with chosen bacteria from its glycerol stock
    3. Incubate and shake at 30 C and take OD600 at 48 hours (Bacteria should be at beginning of lag phase)
  2. Seed sterilization
    1. Add 2mLs of TWEEN 20, 2 mLs of bleach solution, and 8 mls of ddH2O to a 15 ml conical vial to make your seed sterilization solution
    2. Add seeds to sterilization solution and invert for 7 minutes to sterilize
    3. Wash seeds 5x with ddH2O in the same conical vial
    4. Transfer sterilized seeds to an MS agar plate which was wrapped in aluminum foil and incubate at 4°C for four days
  3. Soil preparation
    1. Combine 15g of soil + 10 ml of ddH2O in each 38 x 200mm culture tube needed for your assay
    2. Autoclave soil once per day over 2 days on the liquid cycle (cycle 2) to sterilize
  4. Cell washes and inoculation
    1. Centrifuge for 5 minutes at full speed and discard the supernatant
    2. Wash with 10 ml sterilized saline solution by vortexing
      1. Centrifuge for 5 minutes at full speed
      2. Discard supernatant
      3. Repeat and resuspend with 15 ml sterilized water
    3. Inoculation
      1. Roots: using a syringe transfer 2 ml of final suspension to base of plants. Negative control plants are watered with 2 ml ddH2O.
      2. Seeds: in a sterile petri dish place sterile seeds and soak with remaining culture for 30 minutes at 4°C. Then plant in large cell culture tubes with soil 1 cm under soil surface. Negative control seeds are soaked in ddH2O.
    4. Growth conditions
      1. Grown in a growth chamber at 25°C
      2. Water with 5 ml of sterile water every 3-4 days
    5. Growth time points (taken weekly starting 7 days after inoculation)
      1. Using a sterilized microspatula, loosen soil around the plant
      2. Using a long pair of sterilized tweezers pull up from the base of the plant to extract the plant along with its roots
      3. Place plant inside a sterile petri dish and using a sterilized scalpel cut off the sprout leaving only the roots in the tray.
      4. Wash the roots in the tray with ddH2O then place them on a glass slide. Add ~10 drops of ddH2O to the slide from a dropper and add on a coverslip for confocal microscopy.