17 research outputs found
Stabilisation of a recombinant yeast plasmid in nonselective medium by cyclic growth rate changes
The effect of oxygen limitation on stability of a recombinant plasmid in Saccharomyces cerevisiae
The effect of oxygen limitation on the stability of a recombinant yeast plasmid was measured during continuous culture in a non-selective medium. The fraction of plasmid containing cells was found to decrease more rapidly after a step change to low dissolved oxygen (DOT) levels. An increased rate of plasmid loss appeared to be the major cause of the decreased stability
Effect of growth rate on stability of a recombinant plasmid during continuous culture of Saccharomyces cerevisiae in non-selective medium
The stability of a 2μm based plasmid, pLG669-z, was measured in a Saccharomyces cerevisiae strain, grown continuously in a non-selective medium. The apparent stability of the plasmid was found to decrease with increasing growth rate. A model was developed to predict the contributions of segregational instability and growth rate differences to the apparent stability. An increase in segregational instability was the dominant factor in the increased rate of plasmid loss
A method for the stabilisation of recombinant plasmids in yeast
The stability of a yeast plasmid can be improved using deliberately induced cyclic changes in the dissolved oxygen tension (DOT), during continuous culture in a non-selective, undefined medium. The resultant stability of the plasmid under DOT cycled conditions is strongly dependent on the growth rate of the culture, with complete stabilisation at lower growth rates. We propose a mechanism for the stabilisation and suggest that the method can be applied to other recombinant yeast strains
Stability of recombinant plasmids in yeast
Yeasts represent one class of host for the production of recombinant proteins. Heterologous DNA is usually introduced into yeast strains in the form of multi-copy plasmids. During production, protein expression levels and rates are often limited by the stability of the recombinant organism. In this paper, we review the major factors affecting the stability of yeast strains containing multi-copy recombinant plasmids. Models for predicting plasmid loss are summarised, comparisons are made with relevant bacterial systems and strategies are described for overcoming such problems
Data analysis of plasmid stability in continuous culture of recombinant Saccharomyces cerevisiae
A simple method using non-linear regression is developed to analyse experimental data from plasmid stability studies of recombinant Saccharomyces cerevisiae grown in continuous cultures with non-selective and selective media. This method simultaneously provides quantitative information on the probability of plasmid loss due to segregation during cell division and the specific growth rates of plasmid-containing and plasmid-free cells at particular dilution rates. The method is applied to a set of experimental data. The three-parameter model, together with the estimated parameter values, provides a good fit to the experimental data