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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 14 (1994), S. 131-136 
    ISSN: 1432-203X
    Keywords: Thalictrum minus ; Pectate Lyase ; Berberine ; Stress Metabolite ; Growth Rate ; Phenylalanine Ammonia Lyase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA − E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10−9M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10−11M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 28 (1986), S. 1672-1689 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A structured mathematical model for cellular metabolism in Escherichia coli has been extended to encompass the mechanistic structure surrounding the kinetics and control of transcription and translation. The dependence of transcription on RNA polymerase and the mechanism of translation initiation have been explicitly included. This model correctly simulates cell growth, cell composition, and the timing of chromosome synthesis as a function of extracellular substrate concentration for glucose-limited balanced growth. Simulation results for the subpopulation of RNA polymerase engaged in transcription and for the distribution of this subpopulation among different promoter sites agree closely with experimental findings, as do calculated estimates of the active ribosomal fraction. In addition, the existence of an antitermination system for transcription of stable RNA operons is supported by model results. This model should provide a useful framework for investigating metabolic perturbations to E. coli, such as those resulting from insertion of extra-chromosomal vectors into the cells.
    Additional Material: 9 Ill.
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  • 3
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mechanistically detailed single-cell model E. coli B/r-A was adapted to simulate the effects of vector presence on cell metabolism. Competition for RNA polymerase between chromosome and plasmid DNA is explicitly included. Distribution of active ribosomes among chromosome- and plasmid-derived messenger RNA, another key facet of host-plasmid interactions, is also treated in detail. Simulations of recombinant cell growth rate and cloned-gene productivity as a function of relative plasmid number per cell agree closely with experimental results. Model prediction of the variation of cell cycle parameters C and D with plasmid number are roughly consistent with available data. Models of this class can be used to simulate changes in productivity resulting from specific alterations in the expression vector. The effects of changing cloned-gene promoter and ribosome binding strengths and of augmenting cell transcription or translation capacity have been studied using the recombinant cell model. Results suggest that cloned-gene expression is limited by cellular transcription capacity. These and other parametric studies, conveniently implemented using the computer cell, provide important guidance for future experiments directed at better understanding of host-plasmid interactions and at optimizing recombinant system productivity.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 891-906 
    ISSN: 0006-3592
    Keywords: ribosome vector ; cloned-gene expression ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An expression system utilizing specialized ribosomes has been constructed with β-galactosidase as the product. Ribosomes specific for lacZ mRNA are generated due to a mutation within the anti-Shine-Dalgarno region of a plasmidborne 16S rRNA gene that is complementary to a mutation within the ribosome-binding site of lacZ. Hence, a subpopulation of ribsomes specific for translation of the cloned gene mRNA is produced. Transcription of the lacZ gene is regulated by the tac promoter, while transcription of the mutated rrnB locus is controlled by the λPL promoter. Batch experiments indicate that full induction of both operons (2 mM IPTG, 42°C) leads to maximal β-galactosidase activity per cell at levels 35% higher than that obtained using a wild-type ribosome expression system. Using a novel, site-directed mutagenesis technique, construction of the specialized ribosome vector is outlined, and the results of lacZ expression are presented as transcription of both the cloned-gene and the specialized-ribosome locus are induced.
    Additional Material: 11 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 36 (1990), S. 865-878 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant bacterial systems exhibit limited capacities for heterologous protein production. As seen with this and other systems, cloned-gene protein production reaches an upper limit despite further increases in gene dosage. A series of closely related plasmids which contain mutations affecting their copy number has been used to investigate the macromolecular impediments to enhanced recombinant protein production. Within a common host, HB101, the level of the ampicillin resistance gene, bla, was varied using five plasmids which differ solely in their replication machinery. Separate fermentations were conducted in which the plasmid copy number was varied from 0 to over 400 while the specific growth rate was fixed at 0.6 h-1 for each chemostat cultivation. The effects of constitutive expression of the bla gene as copy number was elevated were then determined using pulse-labelling and RNA-DNA hybridizations. Specifically, the steady-state level, synthesis rate, and stability of β-lactamase messenger RNA and ribosomal RNA were determined as a function of copy number. The results indicate that as copy number rises, both β-lactamase mRNA synthesis rates and steady-state mRNA levels increase. Therefore, β-lactamase production in these strains does not appear to be limited by the level of β-lactamase mRNA. However, as the copy number was amplified, the stability of rRNA decreased to the point that steady-state levels of rRNA decreased. These data indicate that a limitation develops within the translational capacity of the cell at high levels of cloned-gene expression. The results suggest that strategies designed to enhance recombinant protein expression should include manipulation of translation as well as transcription.
    Additional Material: 8 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 329-336 
    ISSN: 0006-3592
    Keywords: biofilm formation ; Escherichia coli ; C/N ratio ; plasmid retention ; extracellular polysaccharide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5α (pMJR1750) were investigated under different medium-carbon-to-nitrogen (C/N) ratios. At C/N ratios of 0.07 and 1, net accumulation of both biofilm plasmid-bearing and plasmid-free cells continued through the entire experiment without attaining any apparent steady state. At C/N ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At C/N ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2g alginate equivalent/cm2 by the end of both experiments. At a C/N ratio of 5, polysaccharide increase significantly after 84 h, reaching about 7μg alginate equivalent/cm2 prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 μg alginate equivalent/cm2 at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 μg/cm2, respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 μg/cm2, respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 205 μg polysaccharide/μg protein, whereas those at C/N ratios of 5 and 10 increased to about 7 and 12 μg polysaccharide/μg protein, respectively.Probabilities of plasmid loss in the biofilm cultures increased with increasing C/N ratios. At C/N ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.0013 ± 0.011, 0.020 ± 0.006 and 0.122 ± 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.38 ± 0.125. The increase of probability of plasmid loss at higher C/N ratios results from competition between cell replication and extracellular polysaccharide production. © 1994 John Wiley & Sons, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 340-352 
    ISSN: 0006-3592
    Keywords: Immobilized cells ; Escherichia coli ; microfluorimetry ; DNA staining ; DNA synthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pronounced spatial nonuniformities in cell density, physiology, and activity frequently arise within densely packed immobilized cell supports. For a more fundamental understanding of immobilized cell phenomena, we have developed high-resolution microfluorimetric procedures to analyze local variations in both immobilized cell loading and growth rate. Fluorescent staining of total cellular DNA provides a measure of local biomass density. Actively growing (DNA synthesizing) cells are marked by pulse-labeling newly synthesized DNA with the thymine analog, bromouracil. An immunofluorescent technique allows subsequent detection of spatial variations in DNA synthesis rates. These procedures enable the influence of mass-transfer limitations and other immobilization effects on cell distribution and activity to be readily quantified. We demonstrate this approach through analysis of the patterns of growth of Escherichia coli entrapped within Sr-alginate gel beads. The experimental techniques are potentially applicable to a variety of other aggregate cell systems.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 397-412 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; protein synthesis ; metabolic limitations ; cloned-gene expression ; β-galactosidase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Earlier experiments in our lab investigated the metabolic limitations of cloned-gene expression in bacterial cells (for over-production of β-lactamase). These experiments showed that the steady-state concentration of ribosomal RNA decreased upon plasmid amplification while both the synthesis rate and steady-state β-lactamase mRNA level increased significantly. This appeared to indicate substantial limitation exist within the transnational machinery of the bacterial cell at high copy numbers. To establish the generality of this phenomenon, the impact increasing protein expression from pa plasmid by chemically inducing a strong promoter while maintaining constant copy number has been investigated. A plasmid has been constructed which contains the lacZ gene under control of the tac promoter and contains the parB stability locus to maintain plasmid stability. Using this vector, β-galactosidase expression in chemostat cultures operated at specific growth rates of 0.6 h-1 was induced with IPTG such that enzyme activity was varied over a 460-fold range. When fully induced β-galactosidase protein production represented 14 wt % of total cell protein. As transcription was induced, the synthesis rate of the β-galactosidase mRNA increased 42-fold while the steady-state level of β-galactosidase mRNA increased only fourfold. This indicates stability may play a larger role for β-galactosidase expression with a strong promoter than seen with β-lactamase production in the elevated copy number system. Furthermore, rRNA synthesis rates increased at high expression rates as seen in the copy number experiments. However, unlike the amplified-plasmid system, the steady-state levels of rRNA increased as well. Since the total protein levels closely followed the steady-state level of eRNA, transnational limitations are again suggested for the chemically induced transcription system.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 211-220 
    ISSN: 0006-3592
    Keywords: plasmid retention ; gene expression ; biofilm ; β-galactosidase ; segregational instability ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5α(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5α(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h-1 to 0.35 h-1 and the β-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h-1, about 36% of that without IPTG, and the β-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5β(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The β-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. © 1993 John Wiley & Sons, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 507-516 
    ISSN: 0006-3592
    Keywords: bioremediation ; Comamonas ; nitrobenzoates ; reactor modeling ; mixed culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model describing the transient activity of a mixed immobilized culture of Comamonas spp. JS46 and JS47 growing on mixed substrates is presented. The transient periods considered are those following changes in the feed carbon source, which alternated between meta- and para-nitrobenzoate. The feed profile alternately starved one of the species in the mixed culture. The response of the system, as quantified by the reactor effluent substrate concentrations, is dictated by the activity of the biomass and the appropriate biochemical pathway. As detailed mechanistic pathway information is not available, respirometry has been used to characterize both facets of activity. Two parameters were introduced: Ψ representing pathway activity and Γ representing biomass activity; a detailed description of the analysis is included. The model is compared to experimental investigation of the system and describes the reactor response well. The agreement between model and experiment suggests the usefulness of oxygen kinetics as global measurements to describe complex systems when mechanistic detail is not available. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 507-516, 1998.
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