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Preparation of Samples for Monod Kinetic Constant Determination

This protocol allows the measurement of the key Monod kinetics Constants based on observing different growth rates for all three auxotrophs individually in various limiting amino acid concentrations.

Day 1

• Set up overnight cultures of the each auxotroph in 5 mL LB with a negative control.

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.
2. Inoculate with each of the three auxotrophs into 200µL M9 culture with various designed corresponding limiting amino acid concentrations and appropriate antibiotics.
3. Set up in a plate reader for >14 hours, measuring the OD600 every 5-20 minutes as intended.

Day 3

• Export the data for the computation of growth rates in all wells of interest, and plot the data to determine the Monod kinetic constants.