Stable maintenance of plasmid in continuous culture of yeast under non-selective conditions

• Published in: Journal of bioscience and bioengineering 2001, Vol.92 (4), p.317-323
• Main Authors: Gupta, J.C. (Jawaharlal Nehru Univ., New Delhi (India)), Mukherjee, K.J
• Format: Article
• Language: English
• Subjects: BETA GALACTOSIDASE; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; PLASMIDS; RECOMBINATION; SACCHAROMYCES CEREVISIAE; STABILITY
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1263/jbb.92.317

A recombinant yeast plasmid containing the gene for beta-galactosidase was tested for stability in a host auxotrophic for leucine. Plasmid loss was studied at different dilution rates in continuous culture under selective as well as non-selective conditions. It was observed that the instability of the culture was higher at low dilution rates in selective medium, while the pattern was reversed when complex non-selective medium was used, with plasmid-containing cells competing effectively with plasmid-free cells at low dilution rates. This was attributed to a low residual yeast extract concentration in the medium at low dilution rates. Since yeast extract was the sole source of leucine, this limited the growth of plasmid-free cells, which were auxotrophic for leucine. Growth rate studies also indicated a competitive advantage of the plasmid-containing cells over the plasmid-free cells at low yeast extract concentrations in semi-defined medium. Using the above data, a modified continuous culture was run using non-selective medium at a low dilution rate of 0.05 h sup(-1). This resulted in stable coexistence of plasmid-containing and plasmid-free cells and hence sustained expression of beta-galactosidase at -330 OD sub420 l sup(-1) h sup(-1) throughout the period of cultivation (134h).