Investigation of subpopulation heterogeneity and plasmid stability in recombinantescherichia coli via a simple segregated model (original) (raw)
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Biotechnology and Bioengineering, 1993
A large number of models concerning cultures of genetically engineered bacteria have been described. Among them, some are specifically adapted to continuous cultures and lead to the determination of two variables: (i) the difference in the specific growth rates between plasmid-carrying cells and plasmid-free cells (Sp) and (ii) the frequency of plasmid loss by plasmid-containing cells (prp+). Until now, studies have been performed on the global expression prp+ and Sp, whose values during continuous assays have been supposed approximately constant (mean value) and not on separate values of both terms p, and p+, respectively, probability of plasmid loss and specific growth rate of the plasmid-carrying cells. So far these studies do not allow examination of the relationship between these two last parameters. Experimental results were obtained with Escherichia coli C600 galK (GAPDH), a genetically engineered strain that synthetizes an elevated quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Biotechnology and Bioengineering, 1989
The instantaneous specific growth rate of a recombinant bacterial culture is directly calculated using a simple structured kinetic modeling approach. Foreign plasmid replication and foreign protein expression represent metabolic burdens to the host cell. The individual effects of these plasmid-mediated activities on the growth rate of plasmid-bearing cells are estimated separately. The dynamic and steady state simulations of the model equations show remarkable agreement with widely observed experimental trends in plasmid copy number and foreign protein content. The model provides an important tool for understanding and controlling plasmid instability in recombinant bacterial fermentations. The modeling framework employed here is suitable for studying the metabolism and growth of a variety of microbial cultures. 12A H1 Atrp :pPLc23 trpAl) and the batch results of Lin-Chao and Bremer" (using E . coli B/rA:pBR322) even though the experimental systems were different.
Stability in continuous cultures of recombinant bacteria: A metabolic approach
Biotechnology Letters, 1990
Continuous-culture population dynamics of recombinant bacteria are predicted with a structured kinetic model. The instantaneous specific growth rates of th.e plasmid-bearing and ptasmidfree cells are explicitly calculated from their metabolic activities. The resultant growth-rate differential (between plasmid-bearing and plasmid-fl'ee cells) is dynamic and changes over the course of a fermentation. Further, the growth-rate differential is a function of dilution rate. We present the experimental determination of model constants governing plasmid replication and foreign protein expression for a host/vector system E. coli RR1 [pBR329]. For a different exper-imental system, we estimate the increased polypeptide expression from. a DNA insert solely from the instability population dynamics. Stability predictions agree quite well with experimental observations from the literature and our tab.
Biotechnology and Bioengineering, 1984
The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of λdv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell. The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids. A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate. Available data, although very limited, show the same trends. These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.
A model for fluctuations in the fraction of a bacterial population harboring plasmids
Theoretical Population Biology, 1986
In some experimental situations the fraction of bacteria containing plasmids appears to repeatedly decline and increase before the plasmids are eventually lost in a gradual terminal decline. These fluctuations may not be observed unless the composition of the population is determined at various cell densities. This paper gives a mathematical model for this behavior. In the model the decreases in the fraction of cells bearing plasmids record the appearance of plasmidless offspring cells of plasmid-bearing cells. The increases occur because plaamidless cells obtain plasmids from multicopy donor cells when the cell density is high. The periodic dilution of the medium necessary to permit continued growth of the population in test tubes repeatedly alters the balance of these two factors. The gradual terminal decline in the fraction of cells bearing plasmids is the result of a slight growth rate advantage of plasmidless cells. During the entire process the average copy number of the cells decreases, and the time lag before the final decline is the time required for the average copy number to approach one.
Microbiology, 1995
The effect of dilution rate on the instability kinetics of Escherichis coli RV308(pHSG415) during glucose-limited continuous culture is examined. Two nonlinear models are fitted to the data, both of which characterize the plasmid-host system in terms of the rate parameters R (for the plasmid segregation rate) and dp (for the specific growth rate difference between plasmid-free and plasmid-bearing single cells). In the first model, both R and dp have constant values with respect to time. In the second, either R or dp is represented as a time-dependent function. Although both models fit the data equally well, it is demonstrated that the constant rate parameter model gives results which appear to be misleading. A comparison is also made among some of the many plasmid instability models (both mass-balance and segregated) which have appeared in the literature. It is found that all of these give identical trajectories and differ only in the definitions of the rate parameters used.
Biotechnology Letters, 1994
The present results concern the recombinant bacteria Escherichia coli HB 101 (GAPDH) which produces glyceraldehyde 3-phosphate dehydrogenase. An unusual phenomenon was noticed concerning the plasmid stability of this strain growing in batch culture. The determination of sensitivity to ampicillin, whose resistance is carried by the plasmid, has been tested as a function of time on Petri dishes containing increasing concentrations of ampicillin in a batch culture in a complex medium without any selection pressure. A transitory decrease in the percentage of resistant cells has been noted during the exponential phase of growth. This phenomenon corresponds to a momentary plasmid instability probably due to a transitory gap between the growth rate of the cell and the duplication rate of the plasmid.
Plasmid maintenance and recombinant cell fitness explored in bacterial colonies
Biotechnology Letters, 2001
During growth of recombinant bacteria, irregular plasmid partitioning generates non-productive, plasmid-free cells whose proportion usually increases in the culture. For Escherichia coli producing engineered β-galactosidases, we have shown a coincidence between plasmid stability and the extension of white/blue areas within individual colonies on X-gal plates. In this context, a good correlation between plasmid permanence in colonies and parameters accurately describing the dynamics of plasmid-free cell population in liquid cultures has been observed. Moreover, the impact of lacZ gene engineering and the metabolic burden imposed by the encoded proteins has been evaluated through plasmid stability by simple image analysis, revealing an enhanced plasmid loss rate as the cells enter into the stationary phase that is modulated by the expression of particular recombinant genes.
Mathematical and Computer Modelling, 2006
The stability of the classical Levin-Stewart model that describes the competition between plasmid-bearing and plasmid-free populations in a chemostat is revisited using a combination of bifurcation theory and continuation techniques. Simple analytical conditions are derived that describe the conditions for the coexistence of the competing cells and for the safe operation of the chemostat. The ability of the model to predict the coexistence of the competing cells in an oscillatory mode is also studied. Analytical results with respect to arbitrary growth kinetics are derived that set the necessary conditions for the existence of Hopf points in the model as well as for the occurrence of a number of Hopf degeneracies. These general conditions are applied to Monod/Haldane substrate inhibition growth models. Practical branch sets in terms of model parameters are readily constructed for Monod-Monod, Monod-inhibition, inhibition-Monod and inhibition-inhibition cases. The dynamic analysis, on the other hand, allows the identification of regions of one and two Hopf points predicted by the model. The combination of results of both static and dynamic bifurcation allows the delineation of a total of 45 qualitatively different regions and helps to construct a useful picture, in the multidimensional parameter space, of the different behaviors predicted by the model. Practical criteria are also set for the comparison between these regions, and for the study of the effects that various limiting substrates can have on recombinant culture stability and as regards the desired rate properties to be looked for in screening media formulations. K. Alhumaizi et al. / Mathematical and Computer Modelling 44 (2006) 342-367 343 (2) instability of the plasmid genetic structure when plasmids are retained in all cells but their forms change, (3) a growth disadvantage of the plasmid-bearing strain compared with the analogous plasmid-free strain variants, and the variants containing plasmids with altered structure.
Modelling, monitoring and control of plasmid bioproduction in Escherichia coli cultures
2012
An integrated approach for modelling, monitoring and control the plasmid bioproduction in Escherichia coli cultures is presented. In a first stage, by the implementation of a kinetic model for E. coli cultures, a better bioprocess understanding was reached, concerning the availability of nutrients and products along the bioprocess, and their effects on the plasmid production. Results presented may provide significant help for future modelling and monitoring implementation. In a second stage, FTIR spectroscopy coupled ...