Team:British Columbia/Protocols/AArate

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This protocol allows the measurement of the production or consumption of amino acids of the corresponding prototrophs and auxotrophs.
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Day 1
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1)Set up overnight cultures of the appropriate producing or consuming strain in 5 mL LB.
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2)Set up overnight cultures of the appropriate testing strain in 5 mL LB.
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Day 2
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1) Wash 1.5 mL producing or consuming culture thoroughly by centrifuging at 10 000 RPM for 1 minutes, removing the LB supernatant, re-suspending in 1.5 mL M9, centrifuging again at 10 000 RPM for one minute, and then repeating the M9 wash at least twice more. Re suspend in 1.5 mL M9.
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2a) For doing production rate experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to 10^-4 molar for optimal growth.
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2b) For doing consumption rate experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to either 10^-4 molar, or a different value for the rate at different initial amino acid concentrations.
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3) Inoculate the cultures with 500 uL re-suspended culture.
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4) Periodically measure OD600 of the culture, and when it reaches the desired OD600, harvest 5mL culture.
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5) Centrifuge down the culture at 10 000 RPM for 5 minutes to remove the cells from the supernatant, and harvest the supernatant. Run the supernatant through a 0.2 uM filter column to remove any other cells.
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6) Repeat for as many OD points as desired. The supernatant can be stored in a 4°C fridge for several days.
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7) Prepare 5X M9 stock (5X sugars+2% glucose), and fill a 96-well plate with 20uL 5X M9. Depending on which cells you are going to inoculate with, you can add other antibiotics. Add 180uL supernatant.
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8a) If doing consumption experiments, inoculate with the testing strain (auxotrophic for the same amino acid as the consuming strain was). Prepare the testing strain inoculate the same way as described in day 2 step 1, and inoculate with 2 uL culture.
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8b) If doing production experiments, inoculate with the testing strain (auxotrophic for as different amino acid than what the producing strain was auxotrophic for). Prepare the testing strain inoculate the same way as described in day 2 step 1, and inoculate with 2 uL culture.
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9) Set up in a plate reader for >14 hours, measuring the OD600 every 15 minutes.
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Day 3
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1) Export data, and compare growth rate and final OD to growth curves of the testing strain under various amounts of amino acids.

Revision as of 07:18, 2 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols

This protocol allows the measurement of the production or consumption of amino acids of the corresponding prototrophs and auxotrophs.

Day 1 1)Set up overnight cultures of the appropriate producing or consuming strain in 5 mL LB. 2)Set up overnight cultures of the appropriate testing strain in 5 mL LB.

Day 2 1) Wash 1.5 mL producing or consuming culture thoroughly by centrifuging at 10 000 RPM for 1 minutes, removing the LB supernatant, re-suspending in 1.5 mL M9, centrifuging again at 10 000 RPM for one minute, and then repeating the M9 wash at least twice more. Re suspend in 1.5 mL M9.

2a) For doing production rate experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to 10^-4 molar for optimal growth.

2b) For doing consumption rate experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to either 10^-4 molar, or a different value for the rate at different initial amino acid concentrations.

3) Inoculate the cultures with 500 uL re-suspended culture.

4) Periodically measure OD600 of the culture, and when it reaches the desired OD600, harvest 5mL culture.

5) Centrifuge down the culture at 10 000 RPM for 5 minutes to remove the cells from the supernatant, and harvest the supernatant. Run the supernatant through a 0.2 uM filter column to remove any other cells.

6) Repeat for as many OD points as desired. The supernatant can be stored in a 4°C fridge for several days.

7) Prepare 5X M9 stock (5X sugars+2% glucose), and fill a 96-well plate with 20uL 5X M9. Depending on which cells you are going to inoculate with, you can add other antibiotics. Add 180uL supernatant.

8a) If doing consumption experiments, inoculate with the testing strain (auxotrophic for the same amino acid as the consuming strain was). Prepare the testing strain inoculate the same way as described in day 2 step 1, and inoculate with 2 uL culture.

8b) If doing production experiments, inoculate with the testing strain (auxotrophic for as different amino acid than what the producing strain was auxotrophic for). Prepare the testing strain inoculate the same way as described in day 2 step 1, and inoculate with 2 uL culture.

9) Set up in a plate reader for >14 hours, measuring the OD600 every 15 minutes.

Day 3 1) Export data, and compare growth rate and final OD to growth curves of the testing strain under various amounts of amino acids.

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