Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture ofEscherichia coli in a non-selective medium (original) (raw)
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Journal of Fermentation and Bioengineering, 1990
The expression of hybrid proteins ,0-galactosidase-human insulin chain A (constitutive), and ,0-galactosidase-human insulin chain A (constitutive), and j0-galactosidase-human insulin chain B (inducible) was studied. The main aspects covered included plasmid stability and optimization of expression levels. In both systems, the ampicillin used for selective pressure exerted its action for only a few minutes during culture, hardly affecting plasmid segregation trends. Without the antibiotic, segregants were very low and reached a level of 10% only after seven sub-cultures. Expression levels in the A chain system were closely related to the biomass production. Maximum levels were reached developing the inoculum in minimal media, as well as by balancing and statistically optimizing the medium. The B chain system appeared to have higher plasmid stability than the A chain one. By optimizing the medium, similar induction levels to those obtained by using IPTG were attained using lactose as the main carbon source. Hybrid production was inversely related to the cell/glucose yield. In both systems, sub-culturing the bacteria in minimal medium increased substantially the production of hybrid proteins. Sub-culturing in rich medium with small amounts of ampicillin had the opposite effect. It seems that plasmid copy number dynamics could be playing an important role in this phenomenon.
Journal of Biotechnology, 1996
Fed batch cultivations of plasmid-free and recombinant Escherichia coli were employed in order to determine cellular responses and effects of plasmid presence and induction on the host cell physiology. While plasmid presence was shown to have minor influence on overall biomass yield, induction with 0.1 mM IFTG led to a marked reduction. The number of dividing cells, measured as colony forming ability, was influenced by plasmid presence and to a larger extent by induction. The latter caused a decline in the number of dividing cells to less than 10% of the population within 10 h. However, this cell segregation did not affect the specific rate of product formation, which was approximately constant throughout the cultivations. Analysis of the in vivo degradation rate of the product indicated that it was proteolytically stable. The cellular content of the stringent response signal substance, ppGpp, peaked immediately after transition from batch to fed batch mode to stabilise at a higher value than in the batch phase. When the specific growth rate declined below 0.06 h-' an additional rise in ppGpp concentration was observed.
Journal of Bioscience and Bioengineering, 2014
Two engineered Escherichia coli strains, designated VH33 and VH34, were compared to their parent strain W3110 in chemostat mode during plasmid DNA (pDNA) production. In strain VH33 the glucose uptake system was modified with the aim of reducing overflow metabolism. The strain VH34 has an additional deletion of the pyruvate kinase A gene (pykA) to increase pDNA formation. pDNA formation rates as well as kinetic and stoichiometric parameters were investigated in dependence of the growth rate within a range from 0.02 to 0.25 h L1. Differences between strains were found in terms of the biomass yields on nitrogen and oxygen, as well as on the cell maintenance coefficients. The deletion of pykA led to a significantly increased pDNA yield and productivity. At an optimal growth rate of 0.20 h L1 it was nearly 60% higher than that of W3110 and VH33. Metabolic fluxes calculated by metabolite balance analysis showed differences mainly in reactions catalyzed by pyruvate kinase and glucose 6-phosphate dehydrogenase. The obtained data are useful for the design of cultivation schemes for pDNA production by E. coli.
Produção e purificação de DNA plasmidial a partir de Escherichia coli recombinante
2003
Cultures of recombinant Escherichia coli containing the plasmid pD2 were grown in two medium TB (Terrific broth) or LBG (Luria Bertani with glucose). Three velocity of agitation (120, 160 and 200 rpm) and five kanamycin concentrations (10, 20, 30, 40 and 50 μg mL ) were the parameters studied for to assess their effects of cultural conditions on plasmid DNA production and stability for plasmid-based gene therapy. The velocity of agitation that provided highest results in relationship biomass and plasmid DNA production was 200 rpm. While the concentrations 30 to 50μg mL of kanamycin provided similar results for all the studied conditions and the culture medium TB medium which showed of better results for the biomass production and DNA. The plasmid was (82%) stable in TB medium.
Effect of growth rate on plasmid maintenance by Escherichia cuZi
The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HBlOl have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-l than at 0.15 h-l. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-l, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene. Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that ,in complex media the plasmid is maintained longer by E. coZi HBlOl at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.
Journal of Biotechnology, 1995
The effect of environmental growth conditions on the segregational stabiIity of a recombinant Esckrichia co&Bacillus subtilis shuttle plasmid pCPPS-31 which expresses carboxymethyl cellulase (CMCase) and contains the neomycin resistance gene (Ne'), was studied in E. coli DHSa. The stability increased with the decrease in medium complexity. Lowering culture temperatures had a negative effect on stability. Stability was maximum at 100 rpm and a medium/flask volume ratio of 1:lO. A pH range of 5-8 had no significant effect on stability. The recombination (ret) and gyrase (gyr) backgrounds of E. coli hosts had no apparent effect on plasmid stability. The plasmid was structurally stable under all circumstances.
Biotechnology and Bioengineering, 1989
Experiments were performed to evaluate, qualitatively and quantitatively, the adaptation of Escherichia coli to plasmid maintenance and cloned gene expression. Experimental findings indicate that the metabolic response to low plasmid levels is an increase of the biosynthetic capacity of both transcription and translation. At high copy number levels the gene-specific transcription rate continues to increase but the stability of plasmid-derived mRNA drops sharply. Protein levels are maintained, but translation efficiency decreases. These results indicate that cellular biosynthetic capacity may not be limiting productivity in recombinant systems. If macromolecular stability is the bottleneck, then current efforts to increase gene expression that focus on enhancing synthesis rates will be ineffective.
Journal of Fermentation and Bioengineering, 1990
The expression of hybrid proteins ,0-galactosidase-human insulin chain A (constitutive), and ,0-galactosidase-human insulin chain A (constitutive), and j0-galactosidase-human insulin chain B (inducible) was studied. The main aspects covered included plasmid stability and optimization of expression levels. In both systems, the ampicillin used for selective pressure exerted its action for only a few minutes during culture, hardly affecting plasmid segregation trends. Without the antibiotic, segregants were very low and reached a level of 10% only after seven sub-cultures. Expression levels in the A chain system were closely related to the biomass production. Maximum levels were reached developing the inoculum in minimal media, as well as by balancing and statistically optimizing the medium. The B chain system appeared to have higher plasmid stability than the A chain one. By optimizing the medium, similar induction levels to those obtained by using IPTG were attained using lactose as the main carbon source. Hybrid production was inversely related to the cell/glucose yield. In both systems, sub-culturing the bacteria in minimal medium increased substantially the production of hybrid proteins. Sub-culturing in rich medium with small amounts of ampicillin had the opposite effect. It seems that plasmid copy number dynamics could be playing an important role in this phenomenon.