Growth of Prototrophic E. coli Strains in D2O Minimal Salts Media

The following is a protocol for growing 1.0 L of E. coli in deuterium oxide (D₂O)-M9 salts medium. Due to the slow growth of cells in D₂O minimal medium, the D₂O culture is inoculated with cells at 0.3 A₆₀₀ grown on H₂O -M9 Salts medium.

To prepare 1L of D₂O medium, follow all the 11 steps listed in the Minimal Salts Media protocol, but a) substitute the water with deuterium oxide (D₂O) in all the solutions prepared, and b) do not autoclave D₂O itself, MgSO₄, glucose, CaCl₂, or yeast extract for sterilization, only filter (0.22mM filter). Also if using ¹⁵N or ¹³C or both, remember to prepare the ¹⁵NH₄Cl or ¹³C-glucose, respectively in D₂O as well.

The protocol below is for culturing cells on 1.0 L of D₂O medium starting from cells growing on H₂O-M9 salts medium:

1. Inoculate 200mL of H₂O-M9 salts medium from fresh colony (as described in Minimal Salts media protocol)

2. Let cells rise to an A₆₀₀ of ~0.3.

3. Centrifuge initial 200mL cells in sterile bottles and dump supernatant.

4. Resuspend pellet in 10mL of D₂O-M9 medium (prepared with ¹⁵N or ¹³C isotope as appropriate).

5. Add the 10mL of resuspended cells to the 1.0 L D₂O culture in sterile flask. This will yield a starting A₆₀₀ of ~ 0.06.

6. Wait for cells to rise at an A₆₀₀ of ~0.7, then induce protein expression with IPTG (or whatever is appropriate) and add 2.5mL of 20% (w/v) D-glucose (or isotope labeled ¹³C-glucose, as appropriate).

7. After induction is complete (typically 5-7 hours), spin down rest of cells 10 minutes at 7500 r.p.m.