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  • 1
    Electronic Resource
    Electronic Resource
    Neither total culture fluorescence nor intracellular fluorescence are indicative of NAD(P)H levels in Escherichia coli MG 1655 (1991)
    Springer
    Applied microbiology and biotechnology 36 (1991), S. 400-403 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The blue fluorescence emitted by microbial cells irradiated with UV light at ∼ 360 nm is usually supposed to provide a good estimate of the cell NAD(P)H content. Here we present an example of a microbial fermentation in which culture fluorescence, both in the cells and in the medium, was almost exclusively due to the presence of a fluorophore that displayed an emission spectrum very similar to that of NAD(P)H but that we show by biochemical studies to be a different compound. Our results demonstrate that studies on the redox state of cells should be based on on-line fluorescence data only after appropriate control experiments to establish a definitive correlation between fluorescence and NAD(P)H levels.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00208164
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  • 2
    Electronic Resource
    Electronic Resource
    Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli (1992)
    Springer
    Applied microbiology and biotechnology 37 (1992), S. 609-614 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Culture conditions favouring the simulataneous formation of soluble protein and inclusion bodies (IBs) were chosen for producing the cytoplasmic protein β-galactosidase or the periplasmic protein TEM-β-lactomase. Soluble and insoluble cell fractions of Escherichia coli producing either β-galactosidase or TEM-β-lactomase were analyzed by one- and two-dimensional gel electrophoresis and subsequent silver staining or immunodection of the recombinant protein. The results show that truncated fragments of the recombinant protein were not present in the soluble cell fraction but accumulate in the IB fraction. The presense of other cellular, non-plasmid-encoded proteins in IB preparations such as the outer membrane proteins OmpF, OmpC, and OmpA or the ribosomal subunit proteins L7/L12 was attributed to co-precipitation of cell-debris-associated components. Protein-folding enzymes were not detected in IB preprations. The specificity of in-vivo protein association in the formation of IBs and its implication on protein purification is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00240735
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  • 3
    Electronic Resource
    Electronic Resource
    Isopropyl-β-d-thiogalactopyranoside influences the metabolism of Escherichia coli (1992)
    Springer
    Applied microbiology and biotechnology 36 (1992), S. 782-784 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a strain of Escherichia coli with one lac promoter in its chromosome causes reduction in synthesis rate for a set of protiens. One of these proteins, designated H35, is a prominent cellular protein present only during exponential growth. Reduction of H35 synthesis is transient and delayed following an IPTG pulse. Cellular response to an IPTG pulse during exponential growth shares several features with a heat shock response. Significant increases in the specific growth rate of cells in both amino-acid-supplemented minimal medium and complex medium were observed for some IPTG concentrations relative to IPTG-free cultures. Other IPTG concentrations caused a reduction in specific growth rate.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00172194
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  • 4
    Electronic Resource
    Electronic Resource
    Proteolytic response to the expression of an abnormal \-galactosidase in Escherichia coli (1992)
    Springer
    Applied microbiology and biotechnology 37 (1992), S. 335-341 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant \-galactosidase was investigated. A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome. Induction of expression of the mutant \-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity. Growth at temperatures above 40°C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37°C, and the ATP-dependent activity increased 2.5-fold from 30 to 42°C. Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal \-galactosidase at 37°C, but no such response was evident when mutant \-galactosidase expression was induced at 30°C. In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid. Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity. These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E. coli. These responses were reduced by induction' at lower temperatures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00210989
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  • 5
    Electronic Resource
    Electronic Resource
    Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α (1989)
    Springer
    Applied microbiology and biotechnology 32 (1989), S. 54-60 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme β-lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5α in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00164823
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  • 6
    Electronic Resource
    Electronic Resource
    The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli (1988)
    Springer
    Molecular genetics and genomics 214 (1988), S. 158-161 
    Details
    ISSN: 1617-4623
    Keywords: Vitreoscilla ; Hemoglobin ; Cloning ; Nucleotide sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3′ end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00340195
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  • 7
    Electronic Resource
    Electronic Resource
    Regulated multicistronic expression technology for mammalian metabolic engineering (1998)
    Springer
    Cytotechnology 28 (1998), S. 111-126 
    Details
    ISSN: 1573-0778
    Keywords: autoregulation ; cell-cycle engineering ; eukaryotic operon ; IRES ; multigene engineering ; picornavirus ; pTRIDENT ; regulated expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008037916674
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  • 8
    Electronic Resource
    Electronic Resource
    pQuattro vectors allow one-step multigene metabolic engineering and auto-selection of quattrocistronic artificial mammalian operons (1998)
    Springer
    Cytotechnology 28 (1998), S. 229-235 
    Details
    ISSN: 1573-0778
    Keywords: CITE ; EMCV ; GFP ; IRES ; picornavirus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Based on internal ribosomal entry sites (IRES) of picornaviral origin we constructed a novel family of mammalian expression vectors. pQuattro vectors contain quattrocistronic artificial eukaryotic operons which link, in a single transcript, the simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest to a terminal neomycin (neo) resistance marker. Due to the strict genetic linkage of the transgenes and the terminal selection marker, this genetic configuration enables, by the selection on neomycin, multigene metabolic engineering of mammalian cells in a single step (one-step metabolic engineering). Furthermore, selection on the terminal cistron of multicistronic expression units enforces cocistronic expression of all upstream encoded genes and maximises genetic integrity of the eukaryotic operon in stable mammalian cell lines, since clones harbouring damaged multicistronic expression units become neomycin-sensitive and are automatically counterselected (auto-selection). The modular set-up and the abundance of restriction sites in pQuattro vectors facilitate the movement of individual genes between multicistronic expression vectors and guarantees high compatibility with genetic elements of a wide variety of existing mammalian expression vectors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008014706196
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  • 9
    Electronic Resource
    Electronic Resource
    A formalism for the solution of problems involving chemical reaction and concentration-dependent diffusion coefficients (1981)
    Springer
    Flow, turbulence and combustion 37 (1981), S. 225-240 
    Details
    ISSN: 1573-1987
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract By using a previously proposed novel integral transform approach, approximate analytical solutions for a certain class of concentration-dependent diffusion and reaction problems can be obtained. Three example problems of increasing complexity are presented to demonstrate the versatility of this technique. Perturbation techniques are also employed to determine asymptotic behaviour of the solutions in the limit of large or small values of some parameters.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00951249
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  • 10
    Electronic Resource
    Electronic Resource
    Streptomycetes in micro-cultures: Growth, production of secondary metabolites, and storage and retrieval in the 96–well format (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 297-305 
    Details
    ISSN: 1572-9699
    Keywords: actinorhodin ; microbial cultivation ; secondary metabolites ; Streptomyces arenae ; Streptomyces coelicolor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mycelium-forming Streptomyces strains were grown in one milliliter liquid micro-cultures in square deep-well microtiter plates. Growth was evaluated with respect to biomass formation and production of secondary metabolites which were found to be very similar in the micro-cultures, bioreactor, and shake flask cultivations, respectively. Despite repetitive sampling and extensive growth on the walls of the wells, no cross contamination occurred. Furthermore, we successfully employed cold storage at −20 °C of spore suspensions (in the 96–well format), directly prepared from cultures grown on agar in the microtitre plate. Cultures were retrieved by replicating aliquots from the frozen spore suspensions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010254013352
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