Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli.

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Kinetic study of pTG201 plasmid stability in Escherichia coli

Recombinant pTG201 plasmid coming from pBR322 plasmid, has been incorporated into Escherichia coli K12 to determine the influence of several culture conditions on the variation of the copy number. Continuous and batch cultures on LB medium without antibiotic selection and different oxygen tensions (21% and 100%) have been tested. The expression of pTGH201 encoded genes and the kinetics of plasmid loss differ significantly from the behaviour of pBR322 plasmid.     

Enhanced plasmid stability and production of hEGF by immobilized recombinant E. coli JM101

Recombinant Escherichia coli JM101 was immobilized with porous polyurethane foam (PUF) particle as supporter matrix for human epidermal growth factor (hEGF) production. Flask culture showed that cell immobilization in PUF can improve cell growth and hEGF expression. A bubble column and a three-phase fluidized bed bioreactor by self-design was further applied to produce hEGF, respectively. The results demonstrated that PUF is a feasible immobilized supporter material with good biocompatibility. Immobilization could also decrease the probability for segregational plasmid loss and overgrowth of plasmid-free cells. Cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. hEGF productivity was enhanced from 8.73 mg/l h of free-culture to 11.4 mg/l h of immobilized cultivation.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Influence of oxygen supply in the production of biomass ofE. coli cells immobilized in K-carrageenan gel

Summary Cells of a recombinantEscherichia coli, immobilized in K-carrageenan, show improved distribution of growth in the gel when cultured in conditions of high oxygen tension. The production of both biomass and protein codified by recombinant DNA is increased.     

Stability without selection pressure of the plasmid pTG 201 during continuous fermentation of Escherichia coli BZ 18 immobilized in a carrageenan gel

Escherichia coli BZ 18 harboring the plasmid pTG 201 and immobilized in carrageenan gel beads in continuous culture without selection pressure, provides a better stability of the plasmid than free cells, with an approximately equal production of biomass.     

Improvement of plasmid stability of recombinant Bacillus-subtilis cells in continuous immobilized cultures

Abstract: The immobilization of recombinant Bacillus subtilis in K-carrageenan gel beads has been performed in order to study the growth conditions inside the gel beads and to improve plasmid stability. Bacterial colonies showing high cell density were studied using scanning electron microscopy. A series of continuous cultures of free and immobilized B. subtilis MT119 (pHV1431, pIL252 and pIL252 Kpn) have been developed without selection pressure. In the free-cell systems, it was found that a loss of plasmid vectors occurred after a short period. In contrast, in the immobilized cell systems, plasmid-free segregants were not detected in any of the cases during the first 80 h of the culture.     

Effects of dissolved oxygen shock on the stability of recombinant Escherichia coli containing plasmid pKN401

The effect of dissolved oxygen shock on the stability of recombinant Escherichia coli cells containing plasmid pKN401 was investigated. The recombinant cells were stable in control batch experiments in media with and without ampicillin. However, these recombinant cells were highly unstable under conditions where a dissolved oxygen shock was induced. The results have implications for design of aerated reactors for recombinant cells.     

Preservation of the beta-galactosidase activity in E. coli cells containing the recombinant pUC19 plasmid

Two BglII fragments of pJC703 cosmid were inserted into the plasmid PUC19. E. coli cells containing the recombinant PUC19/A plasmid acquired rifampicin resistance due to inserted rpoB gene but still expressed the Lac+ phenotype.     

产普鲁兰酶重组大肠杆菌质粒稳定性的研究

通过对产普鲁兰酶的重组大肠杆菌E.coli BL21(DE3)/p ET28a-s-pul菌株在发酵过程中质粒稳定性和普鲁兰酶生成量的考察,发现不同宿主对质粒稳定性及酶活性有重要影响。本文利用E.coli BL21(DE3)p Lys S菌株为宿主,构建重组菌E.coli BL21(DE3)p Lys S/p ET28a-s-pul,通过控制外源蛋白的本底表达,提高了重组菌株的质粒稳定性。优化发酵培养基和发酵条件以后,重组菌产普鲁兰酶能力由480 U/m L提高至627 U/m L,增幅为30.6%。研究结果认为,严格控制外源蛋白的本底表达,是改善重组菌稳定性的重要方法之一。     

Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature-sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.     

Effects of a recombinant gene product and growth conditions on plasmid stability in pectinolytic Escherichia coli cells.

The genes encoding pectin methylesterase (pme) and pectate lyase (pel) from Bacteroides thetaiotaomicron were previously cloned in Escherichia coli. In the absence of selective pressure the recombinant vectors harbouring a functional pel gene were rapidly lost. This instability was due to a toxic effect of the pel gene product when overproduced and was closely related (1) to a decrease of the growth rate, and (2) to the impossibility of transforming different strains of E. coli with the recombinant plasmids harbouring a functional pel gene. When the expression level of the pel gene was reduced and the tet gene partially deleted, the stability was greatly improved. The export of pectate lyase in the extracellular medium was significantly enhanced in the presence of glycine with a positive effect on plasmid stability for low concentrations. Furthermore, using a factorial design at two levels, the effects of tetracycline, ampicillin, glucose and magnesium on pBT4 stability were quantified.     

Improvement in segregational stability of recombinant plasmids by retransformation of E. coli host cells

Summary Strains of E. coli JM103 harboring recombinant pUC8 plasmids were found to exhibit severe segregational instability in the absence of antibiotic selection. This was reversed by retransformation into fresh JM103. pUC8 was considerably more stable than its recombinant derivatives.     

Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli

The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded beta-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased beta-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of beta-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.     

Variation of oxygen requirement with plasmid size in recombinant Escherichia coli

We have previously found an inverse relationship between certain cell growth parameters and plasmid size for a series of recombinant Escherichia coli strains containing pUC8 or one of a series of pUC8 recombinant derivatives. To extend these results we investigated whether there was a similar variation among our strains in oxygen requirement, which might be related to the differences in growth. During logarithmic growth in shake flasks, oxygen uptake by E. coli strain JM103 containing an 8.7-kb pUC8 derivative (pBS5) was 2.5 times that of JM103 harboring pUC8 (2.7 kb) and 7.5 times that of plasmid-free JM103. Supplementing the medium with acetate eliminated both the growth disadvantage of and the increased oxygen uptake by the strain harboring pBS5 compared with that containing pUC8. In all cases oxygen consumption decreased drastically as cells began and then continued into stationary phase, and no significant difference was seen among the three strains at these times. When the three strains were grown in a fermentor with continuous monitoring of oxygen levels, plasmid-free JM103 outgrew JM103 containing pUC8 or pBS5 at three levels of aeration. The latter two strains grew identically when aeration was high; their growth curves diverged, however, when aeration was low. In the fermentor experiments the point at which the growth of the three strains diverged was coincident with the point of oxygen depletion in the cultures.     

Evidence of oxygen limitation in a hollow fibre reactor with immobilized E. coli

Summary A hollow fibre reactor (HFR) has been used to immobilize a regulatory mutant ofE. coli to produce phenylalanine. Fermentation performance in the HFR using both air and pure oxygen shows evidence of significant oxygen limitation. In particular, levels of volatile fatty acids (VFA) produced served as an indication of oxygen limitation.     

The effect of oxygen limitation on stability of a recombinant plasmid inSaccharomyces cerevisiae

Summary 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.