Team:British Columbia/Protocols/AArate

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British Columbia - 2012.igem.org

This protocol allows the measurement of the production or consumption of amino acids of the corresponding prototrophs and auxotrophs, and the generation of calibration curves of the final OD of an auxotrophic strain in varying amounts of amino acids that this measurement requires.

Day 1

1)Set up overnight cultures of the appropriate potential 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 culture of interest thoroughly by centrifuging at 10 000 RPM for 1 minute, removing the LB supernatant, re-suspending in 1.5 mL M9, centrifuging again at 10 000 RPM for 1 minutes, and then repeating the M9 wash at least twice more. Re suspend in 1.5 mL M9.

For measurement of the production or consumption of amnio acids :

2a) For doing potential amino acid production experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to either 5E-5 molar, or a different value for optimal growth.

2b) For doing consumption rate experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to 5E-5 molar, to make sure we can observe obvious differences in final OD reading values later on in the plater reader.

For calibration curve generation:

2c) Skip to step (8c)

3) Inoculate the cultures with 500 µL 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 13 000 RPM for 5 minutes to remove the cells from the supernatant, and harvest the supernatant. Run the supernatant through a 0.2 µm syringe filter 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 40µL 5X M9. Depending on which cells you are going to inoculate with, you can add other antibiotics. Add 160µL filtered 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 µL 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 µL culture.

8c) If doing calibration curve generation, inoculate with the testing strain (all three auxotrophs) into various designed 200µL M9 culture with various designed corresponding limiting amino acid concentrations and appropriate antibiotics.

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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 This protocol allows the measurement of the production or consumption of amino acids of the corresponding prototrophs and auxotrophs, and the generation of calibration curves of the final OD of an auxotrophic strain in varying amounts of amino acids that this measurement requires.
 Day 1
 )Set up overnight cultures of the appropriate potential producing or consuming strain in 5 mL LB.
 )Set up overnight cultures of the appropriate testing strain in 5 mL LB.
 Day 2
 ) Wash 1.5 mL culture of interest thoroughly by centrifuging at 10 000 RPM for 1 minute, removing the LB supernatant, re-suspending in 1.5 mL M9, centrifuging again at 10 000 RPM for 1 minutes, and then repeating the M9 wash at least twice more. Re suspend in 1.5 mL M9.
 For measurement of the production or consumption of amnio acids :
 a) For doing potential amino acid production experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to either 5E-5 molar, or a different value for optimal growth.
 b) For doing consumption rate experiments, add the limiting amino acid and appropriate antibiotic only to 50mL sterile M9 broth to 5E-5 molar, to make sure we can observe obvious differences in final OD reading values later on in the plater reader.
 For calibration curve generation:
 c) Skip to step (8c)
 ) Inoculate the cultures with 500 µL re-suspended culture.
 ) Periodically measure OD600 of the culture, and when it reaches the desired OD600, harvest 5mL culture.
 ) Centrifuge down the culture at 13 000 RPM for 5 minutes to remove the cells from the supernatant, and harvest the supernatant. Run the supernatant through a 0.2 µm syringe filter to remove any other cells.
 ) Repeat for as many OD points as desired. The supernatant can be stored in a 4°C fridge for several days.
 ) Prepare 5X M9 stock (5X sugars+2% glucose), and fill a 96-well plate with 40µL 5X M9. Depending on which cells you are going to inoculate with, you can add other antibiotics. Add 160µL filtered supernatant .
 a) 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 µL culture.
 b) 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 µL culture.
 c) If doing calibration curve generation, inoculate with the testing strain (all three auxotrophs) into various designed 200µL M9 culture with various designed corresponding limiting amino acid concentrations and appropriate antibiotics.
 ) Set up in a plate reader for >14 hours, measuring the OD600 every 15 minutes.
 Day 3
 ) Export data, and compare growth rate and final OD to growth curves of the testing strain under various amounts of amino acids.