Peptone from casein

Cells were grown overnight in shaker TOU-10–2 (MRC Int, Israel) at 200 rpm speed in liquid YPD media (2% glucose (Roth, Germany), 2% peptone from casein (Roth, Germany) and 1% yeast extract (Roth, Germany)) in 30 °C temperature until an optical density of the solution was OD600 = 0.8 − 1.2 (early exponential growth phase). Cell concentration was 4–9 × 10⁷ cells/mL. Optical density was measured using Halo-RB-10 (Dynamica Scientific Ltd., GB) spectrophotometer. Then cells were centrifuged at 5000 rpm (2000 g) for 4 min and resuspended at room temperature in electroporation buffer EPB (20 mM Tris (AppliChem, Germany), 1 M sorbitol (Fisher Scientific, USA), pH 7.4) at a volume rate of 1:1. Centrifugation and resuspension procedure was repeated 3 times. During the experiments cell suspensions were stored on ice.

As complex medium LB medium was used for E. coli pre-cultures containing 10 g/L peptone from casein (Carl Roth, Karlsruhe/Germany), 5 g/L yeast powder extract for bacteriology (Carl Roth, Karlsruhe/Germany) and 5 g/L NaCl. The pH value was adjusted to pH 7.0–7.2. 50 mg/L kanamycin sulfate was added to maintain selection pressure.
All cultivation experiments of E. coli were performed in the modified Wilms-MOPS mineral medium [52 (link), 56 (link)] with the base solution consisting of 6.98 g/L (NH₄)₂SO₄, 3 g/L K₂HPO₄, 2 g/L Na₂SO₄, 41.85 g/L (N-morpholino)-propane sulfonic acid (MOPS). The pH value was adjusted to 7.5 with NaOH. Before each experiment, the base solution was supplemented with 0.5 g/L MgSO₄·7H₂O, 0.01 g/L thiamine hydrochloride, 1 mL/L trace element solution [0.54 mg/L ZnSO₄·7H₂O, 0.48 mg/L CuSO₄·5H₂O, 0.3 mg/L MnSO₄·H₂O, 0.54 mg/L CoCl₂·6H₂O, 41.76 mg/L FeCl₃·6H₂O, 1.98 mg/L CaCl₂·2H₂O, 33.4 mg/L Na₂EDTA (Titriplex III)] and 50 mg/L kanamycin sulphate to maintain selection pressure.
The culture medium of batch cultivations was supplemented with a glucose solution to achieve a final concentration of 20 g/L glucose. In the culture medium of fed-batch cultivations, sterile distilled water was used to compensate the omitted glucose volume to maintain the same composition of the other medium components.

The cell suspension was diluted by performing serial dilutions until overall dilution was 8000 times and plated on solid YPD media (2% glucose (Roth, Germany), 2% peptone from casein (Roth, Germany) and 1% yeast extract (Roth, Germany), 1.2% agar (Merck, Germany)). Plates were incubated for 48 h at 30 °C temperature in INCU-Line (VWR, USA) incubator. After incubation, the number of colony-forming units (CFU) was counted. A viability of 100% corresponds to the number of CFU formed by untreated cell suspension.