Use of Continuous Culture for the Selection of Plasmids with Improved Segregational Stability (original) (raw)
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Stable Maintenance of Plasmid in Continuous Culture of Yeast under Non-Selective Conditions
Journal of Bioscience and Bioengineering, 2001
A recombinant yeast plasmid containing the gene for /3-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 leutine, 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-l. This resulted in stable coexistence of plasmid-containing and plasmid-free cells and hence sustained expression of P-galactosidase at -330 OD,,, t' h-' throughout the period of cultivation (134 h).
On the stability of plasmid DNA vectors during cell culture and purification
Molecular Biotechnology, 2007
Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4°C, >12 weeks at -20°C) prior to processing. With alkaline lysates, however, storage at -20°C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization.
Biotechnology Letters, 1992
Experimental results were obtained with Escherichia coli C600 galK(GAPDH), a genetically engineered strain that synthetizes a large quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (80 % of the soluble proteins). Data concerning the stability of plasmid-containing cells and gene expression as a function of dilution rate have been obtained in continuous cultures. Contrary to other studies, our results show a clear indication that the rate of the recombinant activity was dependent on dilution rate. The results support the finding that the apparent stability of the plasmid decreases with dilution rate.
Journal of Biotechnology, 1999
A recombinant strain of Saccharomyces cere6isiae containing a plasmid-encoded lacZ gene from Escherichia coli was grown for 420 generations under selective conditions in glucose-limited continuous culture. A ura3-based auxotrophic system was used to apply selection in favour of plasmid-containing organisms. A similar strategy had previously proved successful at evolving clones of Bacillus subtilis, showing improved plasmid stability characteristics. In this study a series of clones were isolated which exhibited large variation in their ability to retain the recombinant plasmid. Clones showed both significantly increased and reduced capacity to maintain the recombinant plasmid. The probabilities of obtaining clones in either category were essentially equal so that selection was not seen to enrich for more stable clones. Periodic selection events appeared to exert a greater influence on the distribution of stability characteristics amongst clones than did the applied selective pressure. Alterations in plasmid retention characteristics could be associated with host or plasmid. The most stable clone isolated exhibited a 30% improvement of its overall stability (s(N +)) and an 80% improvement in productivity, when compared to the parental strain CGpLG. This improved stability was associated with alterations in the plasmid genome.
Applied Microbiology and Biotechnology, 1992
A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coll. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.