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  • 1
    Electronic Resource
    Electronic Resource
    An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 1027-1038 
    Details
    ISSN: 0006-3592
    Keywords: continuous fermentation ; pili ; plasmid segregation ; gene expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth-rate differential between plasmid-bearing and plasmid-free cells, and thereby affects the culture dynamics of a plasmid-containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl tranferase (CAT). Experimental and theoretical results show that plasmid-bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild-type levels, and thus have more of a metabolic burden than do the plasmid-bearng cells that overproduce CAT. However, when the level of the plasmid-directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid-bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid-containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments. © 1992 John Wiley & Sons, Inc.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260400906
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  • 2
    Electronic Resource
    Electronic Resource
    A novel continuous centrifugal bioreactor for high-density cultivation of mammalian and microbial cells (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 1190-1202 
    Details
    ISSN: 0006-3592
    Keywords: continuous centrifugal bioreactor ; ethanol fermentation ; mammalian cell hybridoma ; monoclonal antibody ; fluidized bed ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A continuous centrifugal bioreactor (CCBR), developed to study the growth and productivity of dense suspensions cultures, has been applied to both fermentation and mammalian cell cultivation processes. With this approach, high-density nonflocculent cultures are maintained in a tapered fluidized bed by balancing the drag forces on the cells due to following substrate with the centrifugal forces. The Sysyem was first used to produce ethanol by fermentation with Saccharomyces cerevisiae; then with H21A1 mouse hybridoma cells secreting monoclonal antibody (MoAb), lgM. Results of this research show the feasibility of using the CCBR for both production of secreted products and as a research tool for studying cell metabolism and production kinetics. Media recycle may be used to modify the behavior of the system form a plug flow apparatus to a continuous stirred reactor (CSTR).
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260381011
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  • 3
    Electronic Resource
    Electronic Resource
    Dynamics of induced CAT expression in E. coli (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 749-760 
    Details
    ISSN: 0006-3592
    Keywords: metabolic modeling ; bioreactor optimization ; recombinant protein synthesis ; induction dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The dynamics of chemically induced chloramphenicolaceyl-transferase (CAT) expression are determined in batch cultures of Escherichia coli DH5αF'IQ [pKK262-1]. This article is directed towards understanding the coupling of induced cloned-protein synthesis and reduced cell growth which are balanced in the optimal system. Experimental results indicate that the best inducer (IPTG) concentration is near 1.0 mM when added during midexponential growth. Lower concentrations cause only weak induction, whereas higher concentrations cause sufficiently strong induction that cell growth is suppressed. Induction at the onset of the stationary phase results in high expression but is accompanied by stimulated protease activity. Also, cell mass yield is adversely affected by enhanced protein synthesis. A structured metabolic model is shown to predict the responses of instantaneous growth rate and productivity which result from protein overexpression. This model can be employed to predict alternative reactor strategies and operating conditions necessary for the design of efficient bioprocess.
    Additional Material: 15 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260380709
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  • 4
    Electronic Resource
    Electronic Resource
    Plasmid-encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 35 (1990), S. 668-681 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experimental elucidation of the metabolic load placed on bacteria by the expression of foreign protein is presented. The host/vector system is Escherichia coli RR1/pBR329 (ampr, camr, and letr). Plasmid content results, which indicate that the plasmid copy number monotonically increases with decreasing growth rate, are consistent with the literature on ColE1-like plasmids. More significantly, we have experimentally quantified the reduction in growth rate brought about by the expression of chloramphenicol-acetyl-transferase (CAT) and β-lactamase. Results indicate a nearly linear decrease in growth rate with increasing foreign protein content. Also, the change in growth rate due to foreign protein expression depends on the growth rate of the cells. The observed linear relationship is media independent and, to our knowledge, previously undocumented. Furthermore, the induction of CAT, mediated by the presence of chloramphenicol, is shown to occur only at low growth rates, which further increases the metabolic load.Results are vdelineated with the aid of a structured kinetic model representing the metabolism of recombinant E. coli. In this article, several previous hypotheses and model predictions are justified and validated. This work provides an important step in the development of comprehensive, methabolically-structured, kinetic models capable of prediciting optimal conditions for maximizing product yield.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260350704
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  • 5
    Electronic Resource
    Electronic Resource
    Plasmid maintenance and protein overproduction in selective recycle bioreactors (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 37 (1991), S. 325-333 
    Details
    ISSN: 0006-3592
    Keywords: segregation ; flocculation ; induction ; fermentations, continuous ; protein overproduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein β-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260370406
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  • 6
    Electronic Resource
    Electronic Resource
    Separations of derivatized amino acid enantiomers by cyclodextrin-modified capillary electrophoresis: Mechanistic and molecular modeling studies (1994)
    Weinheim : Wiley-Blackwell
    Electrophoresis 15 (1994), S. 785-792 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The enantiomers of 5-dimethylamino-1-naphthalene sulfonyl (DNS)-deriva-tives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio-selective running buffer additives. A previously described model for retention and chiral recognition in CD-modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid-CD complexation constants for enantiomer pairs. In this work, the D-enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co-elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS-amino acid enantiomer-CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer-CD interaction energies is shown to provide reasonable correlation with separation performance.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501501110
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