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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 697-704 
    ISSN: 0006-3592
    Keywords: lambda phage ; lysogeny ; lytic state ; partial lysis ; multiplicity of infection ; temperature induction ; intracellular ; extracellular product ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The integrated state of λ in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on λ expression systems21,22 have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of λ systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential λ protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector λZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in λ systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of λ vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. © 1992 John Wiley & Sons, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 280-286 
    ISSN: 0006-3592
    Keywords: biofilm ; plasmid transfer ; conjugation ; mathematical models ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 280-286, 1998.
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  • 3
    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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  • 4
    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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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 272-279 
    ISSN: 0006-3592
    Keywords: biofilm ; plasmid transfer ; conjugation ; retrotransfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 272-279, 1998.
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  • 6
    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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  • 7
    ISSN: 0006-3592
    Keywords: Comamonas ; nitrobenzoates ; bioremediation ; immobilized mixed culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The treatment of meta- and para-nitrobenzoic acid in an industrial wastestream by Comamonas sp. JS46 and Comamonas sp. JS47 is investigated. The most important feature of the wastestream is the constantly changing concentration ratio of the two isomers. The most extreme occurrence is considered here: the complete change in feed carbon source from one isomer to the other. A series of immobilized cell airlift reactor experiments are described to examine the operation and response of the system to these changes in the feed carbon source. Separate reactors containing each species immobilized are compared with a reactor containing both species immobilized within the same bead, and to a reactor containing both species with each species confined to separate beads. On the basis of response time necessary to recover the appropriate activity, the reactor containing both species immobilized within the same bead offers the most effective arrangement. Interactions occurring between the two organisms in the coimmobilized system, mediated by the nitrobenzoate metabolites, are discussed relative to the improved response of this arrangement. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:21-27, 1998.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 1-12 
    ISSN: 0006-3592
    Keywords: Pseudomonas ; substrate inhibition ; metabolic flux ; pathways analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inhibition by toxic substrates enables multiple steady states to arise in biodegradation systems. This phenomenon was investigated for the continuous metabolism of aniline by Pseudomonas sp. CIT1. Differences of various metabolic parameters between the two growth regimes (uninhibited and inhibited) and the transient response to a step-up in dilution rate were determined. Regulatory mechanisms consistent with the experimental evidence are proposed.Aniline is the transcriptional inducer of a metabolic pathway that converts aniline to TCA cycle intermediates. The suite of enzymes is coordinately expressed from a single promoter. We followed the level of the pathway mRNA using a fragment containing the catechol 2,3 dioxygenase gene (andioxB) and monitored the pathway enzyme activity using catechol 2,3 dioxygenase (C23D). The inhibited regime resulted in a 60% lower growth yield, near constant levels of C23D monomer, but a 50% reduction in the specific activity of C23D, increased RNA synthesis rates (total and aniline pathway mRNA), and elevated RNA decay rates.Elucidation of regulatory mechanisms indicates that C23D is noncompetitively inhibited by aniline and subject to feedback inhibition by 2-hydroxymuconic semialdehyde (HMS). During uninhibited growth regime operation, metabolism of HMS is the rate-limiting step; in contrast, conversion of aniline to catechol limits growth in the inhibited regime. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:1-12, 1998.
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  • 9
    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.
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  • 10
    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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