High-Cell-Density Culture to Produce Plasmid DNA for Gene Therapy in E. coli - How to produce Plasmid DNA in a high-cell-density culture. - BioPharm International

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High-Cell-Density Culture to Produce Plasmid DNA for Gene Therapy in E. coli

Culture Conditions

Shake-flask cultures were grown in 500 mL of LB medium and agitated at 200 rpm in a rotary shaking incubator for 6 h at 37 °C. Fed batch fermentations were aerated at 5 L/min and stirred at 400–700 rpm to maintain an oxygen saturation rate of 20%. The pH level was maintained at 7.0 ± 0.2 by automatic additions of 25% (v/v) NH3 and 85% (v/v) phosphoric acid. Foaming was controlled automatically by the addition of an antifoaming agent.

Dry Cell Weight Determination

To determine the dry-cell-weight, 1 mL aliquots of fermentation culture were centrifuged at 10,000 rpm for 10 min in preweighed plastic tubes. After careful removal of the supernatant, cells were resuspended in an equal volume of sterile purified water and centrifuged under the same conditions. The supernatant was decanted and the cell pellets were dried to constant weight at 105 °C.

Glucose and Total Protein Assay

Glucose concentration in the supernatant was determined by the dinitrosalicylic acid reducing-sugar assay.11 Total protein concentration was measured using the Lowry method.

Plasmid DNA Isolation

Each replicate of the 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 using the alkaline method.12

Gel Electrophoresis

Agarose gels (0.8%) were prepared 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. The photographs were taken using a video camera (GelPrinter Plus, TDI, Madrid, Spain).

Analytical Method for Quantification

Absorbance of the DNA samples at 260 and 280 nm was measured using an HP spectrophotometer. The concentration of plasmid DNA was calculated from the A260 data. The purity of the samples was checked by the ratio of absorbance at 260 nm and 280 nm.

RESULTS AND DISCUSSION

Design of the High-Cell-Density Culture Medium

Media composition can dramatically affect plasmid quality and yield. A high-cell-density fermentation requires a balanced medium supplying adequate amounts of nutrients for energy, biomass, and cell maintenance, and commonly contains carbon and nitrogen sources, various salts, and trace metals. Taking these requirements into consideration, we designed a culture medium with a bacterial element composition similar to what has been reported in the literature.13


Figure 1. Cell growth kinetics of recombinant E. coli DH10B transformed with pIDKE2 in the designed medium in a 5-L fed batch fermentation process
The results displayed in Figure 1 show that when cells were grown in this medium, cell growth was in the log phase from hour 1 to approximately hour 21, and in the stationary phase from approximately hour 22 to 25. Cell density then began to decrease at approximately hour 25 or 26.

Carbon source exhaustion occurred after 5 h of batch growth, as indicated by the increase in pH (>7.2), which was caused by the consumption of the alternative carbon source and confirmed by a reducing-sugar assay. Starting precisely at that moment, a mixture of glucose and yeast extract was fed at a constant flow rate and glucose accumulation was observed only when feeding started.


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