The recombinant host Escherichia coli DH10B bearing the plasmid pIDKE2 was grown under fed batch conditions, and the effects of different medium components on plasmid yield and cell mass were evaluated at a 5-L fermentation scale. Results showed that glucose was the optimal carbon source at 265 g/L. After testing different levels of nitrogen, a defined complex medium was formulated for optimal plasmid production, capable of producing up to 0.44 mg plasmid DNA per g dry cell weight.
A further feature of fermentation processes for large-scale plasmid production is the performance of high-density fermentations to obtain large amounts of biomass. Experimental work on the composition of bacterial growth media has demonstrated that fermentation conditions and growth media strongly influence the yield and quality of plasmids produced in E. coli cells.9
In this work, the medium composition of a high-cell-density culture for the production of plasmid pIDKE2 in a recombinant E. coli DH10B system was studied. The results provide a defined medium for pDNA production.
MATERIALS AND METHODS
Strain and Plasmid
The plasmid used in this work was pIDKE2 with a kanamycin-resistant marker; it was transfected into E. coli DH10B using standard methods.1 The E. coli was then grown on several LB agar plates supplemented with 50 μg kanamycin/mL.
The basic LB medium for seed cultivation contained 10 g tryptone/L, 5 g yeast extract/L, and 10 g NaCl/L, supplemented with 50 μg kanamycin/mL. A fed-batch fermentation was carried out in a 5-L bioreactor with a working volume of 4 L, in a complex medium containing 5 g glucose/L, 1.4 g KH2PO4/L, 8.6 g (NH4)2SO4/L, 1 g MgSO47H2O/L, 10 g yeast extract/L, 1 mL trace metal solution 1,000 X/L, and 50 μg kanamycin/mL. The trace metal solution 1X was prepared as follows: 2.29 mg AlCl3·6H2O/L, 1.6 mg (CoCl2·6H2O)/L, 41.21 mg H3BO3/L, 4.98 mg (MnSO4·H2O)/L, 0.73 mg (Na2MoO4·2H2O)/L, 13.7 mg (CuSO4·H2O)/L. Using a peristaltic pump, feed medium (265 g glucose/L and 50 g yeast extract/L) was added to the fed batch cultures at a constant flow rate of 0.7 mL/min when the pH had risen, because that was a signal that the initial carbon source had been depleted.