Genome Minimization Growth / Death Assays

In order to be able to compare parameters uniformly between evolved and ancestor strains, all growth in these tests is done at 200 µM thymidine (dT). This will not detect adaptation to growth at lower dT concentrations.

Day -2 Revive

Revive strains to be tested by inoculating 10 ml of DM0+0.2% glucose+200 µM dT with 100 µl (for a mixed population) or 10 µl (for a clone) in a 50 ml flask.

Grow 16-24 hours at 37°C shaking at 120 rpm.

Day -1 Precondition

Transfer 100 µl of each culture to 10 ml of fresh DM0+0.2% glucose+200 µM dT in a 50 ml Erlenmeyer flask.

Grow exactly 24 hours at 37°C shaking at 120 rpm.

Day 0 Wash and Begin Growth / Death

Transfer entire 10 ml of culture into a 15 ml conical orange-capped tube. Centrifuge for 4 minutes at 6,000 rpm. Decant supernatant. Wash cell pellet by adding 10 ml of DM0 and vortexing until cells are resuspended. Repeat centrifuge and washing step. Centrifuge one final time and resuspend in 10 ml of DM0.

Transfer 100 µl of washed cells to DM0 (no growth/death control), DM0+0.2% glucose (death), DM0+0.2% glucose+200µM dT (growth). The initial cell density will be approximately 4 x 10⁷ cells / ml.

In the control, the cell number should remain nearly constant for at least 48 hours.

In the death cultures, the cell number will decrease after a lag of 30-40 minutes.

Make 10-fold serial dilutions by arraying 90 µl spots on an empty petri dish with a multichannel pipettor. Remove 10 µl of each culture and mix it with the first spot. Plate 10 µl of this mixture, then transfer 10 µl to the next 90 µl spot. Plate 10 µl of this on an MG+dT or TA+dT plate. Repeat dilution and plating until the desired concentration range has been covered.