Team:Washington/Protocols/EG Assay


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Turbidostat: Ethylene Glycol Assay

  1. Turbidostat Preparation
    1. Created M9 30mM ethylene glycol media
      • Sterile 200mL of 5X M9 salts
      • 2.8mL of filter sterilized 99.99% ethylene glycol
      • 800mL of sterile double distilled water
    2. Autoclave the closed system of tubing, syringes, culture vessel, and empty bottle
      • Wrap all open ends of the system with aluminium foil before autoclaving to prevent autoclave water from entering the closed tubing system
    3. Place closed system into the turbidostat apparatus
    4. Carefully and sterilely decant previously made sterile M9 30mM ethylene glycol media into the media bottle
    5. Place the turbidostat into a 37º C incubator
    6. Connect the turbidostat to a computer with the turbidostat software and related libraries installed
    7. Start up turbidostat software program
      • Be sure to close incubator door to reduce amount of ambient light
    8. Wait till program reads out optical density values - should see "0.000" as first optical density value
  2. Cell Preparation
    1. Culture an overnight of MG1655 transformed with fucO pGA3K3 and aldA pGA1C3 in 2mL of TB with kanamycin and chloramphenicol
    2. Take 1mL from the overnight culture and pipette it into a 1.5mL microcentrifuge tube
    3. Pellet the 1mL aliquot at 4000g for 3 minutes
    4. Pour out the supernatant
    5. Resuspend the pelleted cells with 1mL of sterile PBS
    6. Repeat steps 3-5 two more times
  3. Turbidostat Inoculation and Operation
    1. Take a hypodermic needle attached to a 2.5mL syringe and aspirate 0.5mL of washed cells
    2. Open incubator and through the soft stopper at the top of the culture vessel, inject the cells into the culture vessel to an OD ~0.2
    3. Close incubator door
    4. Wait 12-24 hours
    5. Stop turbidostat program
    6. Retrieve data files from program folder and analyze them using your favorite mathematical software (ie: Matlab or Mathematica)