Studies On Fermentation And Purification Processes Of Plasmid DNA

By using the plasmid pcDNA3 containing strain E.coli DH5αas a material, this thesis systematically studied on the processes of fermentation, cell lysis, and anion-exchange chromatography, which are designed for the large-scale production and purification of plasmid DNA. First, an orthogonal study was performed to investigate influences that the chosen factors and their different levels imposed on the cell density of the culture. According to the results, a series of optimal parameters that are suitable for the high- density fermentation of bacteria were selected. Meanwhile, experiments of determining the growth curve of the bacteria and purifying the plasmids were carried out simultaneously to study the relationship between the amounts of the plasmids in the culture and the growth curve, and find out the favorable time for harvesting the plasmids.Secondly, large-scale fermentation processes including batch and fed-batch had been studied in detail. The relationship of biomass growth and concentration of plasmid was investigated.Finally, alkaline lysis and anion exchange chromatography were used to release and purify plasmids. By changing the conditions of chromatography, for example, the matrix, the composition of the mobile phase, the methods of elution, the loading volume, and so on, varied results are acquired, and an optimal method was determined for the purification of plasmids by anion exchange chromatography at last.