1. Cultivation of Escherichia coli

The MRE600 strain of E. coli bacteria was used. The main reason for this is its good tolerance to 100% D₂O during growth (Vanatalu et al., 1993). In terms of the procedures to grow these cells, the fed-batch approach or HCDC (high cell density cultivation) was used to enhance biomass production (Riesenberg et al., 1990). This procedure has exploited modern fermenter systems (Labfors sytems, Infors).

2. Adaptation process

Growing E. coli MRE600 bacteria in a deuterated medium requires a process of adaptation, summarised below.

Figure 1 : The procedure developed for the adaptation of E. coli MRE600 Cells to deuterated media.

The Enfors derivative medium (Enfors S.O. et al., 2000) :

For 1 liter,
6.86 g (NH₄)₂SO₄	*Metal salts (for 1 liter)
1.56 g KH₂PO₄	0.5 g CaCl₂.2H₂O
6.48 g Na₂HPO₄.2H₂O	16.7 g FeCl₃.6H₂O
0.49 g (NH₄)₂-H-citrate	0.18 g ZnSO₄.7H₂O
5 g deuterated glycerol-D8	0.16 g CuSO₄.5H₂O
Sterilise by autoclaving and then add	0.15 g MnSO₄.4H₂O
1 ml MgSO₄ 1M (sterilised by filtration)	0.18 g CoCl₂.6H₂O
1 ml Metal salts*	20.1 g Na-EDTA
	Sterilised by filtration

The deuteration of the inorganic materials is achieved by flash evaporation : each hydrated component is dissolved in the minimal required volume of D₂O and the solution is then evaporated at 60°C with the use of a rotavapor.

3. High Cell Density Cultivation

MRE600 cells are first grown in flasks at 37°C until an OD600 of about 2 is obtained. The culture is then used to inoculate a 2 liter-fermenter at 5-10%. In the fermenter, cells are grown in a medium containing 5 g/l of carbon source. Fermentation parameters (e.g. stirrer speed, temperature, pH, pO2 and gas flow rate) are simultaneously controlled. Nutrient feeding (solution of deuterated glycerol-D8 at 8%) is then added, with an increasing feed rate, in the fed-batch stage.

At the end of the fermentation, cells are harvested by centrifugation at 6Krpm for 30 min, 4°C. Cells are then frozen in liquid nitrogen and stored at –80°C. Fermentation yields approximately 40 gms of deuterated MRE600 cells by wet weight per liter.

Figure 2 : Profile of a typical fermentation in a deuterated medium.

4. Purification of perdeuterated chromosomal DNA from deuterated MRE600 cells

The extraction and purification of perdeuterated chromosomal DNA follows essentially the same recipe as used for hydrogenated DNA, although additional steps have been introduced to optimise the efficiency of the process. Cells are suspended, lysed, and centrifuged. The resulting material is resuspended, treated with RNAse, and subjected to repeated phenol/chloroform purification cycles, and ethanol precipitation. The pure material is then dialysed against TNE and either stored in solution or lyophilised and kept for future use.

For further information, contact Ingrid Parrot (parrot@ill.fr)