No abstract

OATAO is an open access repository that collects the work of some Toulouse researchers and makes it freely available over the web where possible.

No abstract

The recovery of plasmid DNA from cells is achieved by a two-step chemical reaction which usually employs sodium hydroxide and sodium dodecyl sulphate followed by neutralisation with a chilled solution of potassium acetate. It is important that the gelatinous¯oc of chromosomal DNA with proteins debris is not broken down since fragments of chromosomal DNA are dif®cult to separate from plasmid DNA. To accomplish the operation at scale demands knowledge of the rheology as the reactions proceed. In this paper a co-axial cylinder rheometer is used to record the changes for two strains of Escherichia coli cells. Analysis of the liquor, prior to neutralisation indicates that at shear rates below 367 s A1 the¯ow properties during lysis are non-Newtonian, but above 367 s A1¯o w behaviour becomes Newtonian. Additionally, following neutralisation, the rheological data show that the clear liquor obtained at low shear levels is Newtonian, but the gelatinous¯oc has strong viscoelasticity. These properties strongly in¯uence subsequent stages for the removal of solids.

Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5alpha/pSVbeta was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s(-1), with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed.

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.