MATERIALS AND METHODS

Strain and plasmid

The plasmid used in this work was pIDKE2 with a Kanamicin resistance marker. It was transformed into E. coli DH10B host cells as described (11). The transformed E. coli was grown on several LB agar plates supplemented with 50 μg Kanamicin/mL.

The basic LB medium for seed cultivation contained (per L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, supplemented with 50 μg Kanamicin per mL. A fed-batch fermentation was carried out in 5-L and 50-L bioreactors in a working volume of 4 L and 40 L respectively, with complex medium containing (per L): 5 g glucose, 1.4 g KH₂PO₄, 8.6 g (NH₄)₂SO₄, 1 g MgSO₄ ·7H₂O, 10 g yeast extract, 1 mL trace metal solution 1000X, and 50 μg Kanamicin per mL. The trace metal solution 1X was prepared using the following (per L): 2.29 mg AlCl₃·6H₂O, 1.6 mg CoCl₂·6H₂O, 41.21 mg H₃BO₃, 4.98 mg MnSO₄·H₂O, 0.73 mg Na₂ MoO₄·2H₂O, 13.7 mg CuSO₄·H₂O. The addition of feed medium (263 g glucose/L and 50g yeast extract/L) to fed-batch cultures at a constant flow rate of 7 mL/min was achieved using a peristaltic pump when the initial carbon source was deployed due to an increase of pH.

Culture conditions

Shake-flask cultures were grown in 500 mL of LB medium, and were agitated at 200 rpm in a rotary shaking incubator for 6 hours at 37 °C. Fed-batch fermentations were aerated at 25 L/min and stirred at 385 rpm to keep 20% saturation of oxygen levels. PH was maintained at 7.0 ± 0.2 by the automatic additions of 25% (v/v) NH₃ and 85% (v/v) phosphoric acid. Foaming was controlled automatically by the addition of antifoaming agent.

Dry-cell-weight determination

For dry-cell-weight determination, 1 mL aliquots of fermentation culture were centrifuged at 10,000 rpm for 10 min in a preweighed plastic tube. After careful removal of the supernatant, cells were resuspended in an equal volume of sterile pure H₂O and centrifuged. The supernatant was decanted and the cell pellets were dried to a constant weight at 105 °C.

Glucose and total protein assay

Glucose concentration in supernatants was determined by dinitrosalicylic acid reducing-sugar assay and total protein concentration was determined using the Lowry method (12).

Plasmid DNA isolation

Each replicate of 10 mg bacterial cell pellet was resuspended in 300 μl of 50 mM Tris-HCl, 10 mM EDTA, 100 μg RNase A/mL, pH 8.0, and purified by the alkaline method (13).

Gel electrophoresis

Agarose gels (0.8 %) were made up in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) containing 0.5 μg ethidium bromide/mL. Gels were run at 100 V (5 V/cm), for 1 h. Photographs were taken using a video camera (GelPrinter Plus, TDI, Madrid, Spain).

Analytical method for quantification

Absorbance of DNA sample at 260 nm and 280 nm was determined with an HP spectrophotometer. Concentration of plasmid DNA was calculated from A260. The purity of samples was checked by the ratio of absorbance at 260 nm and 280 nm.