The use of lac-type promoters in control analysis

Peter Ruhdal Jensen, H. v. Westerhoff, Ole Michelsen

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady-state fluxes or concentrations. We describe an improved method for effecting the modulation, as elaborated for Escherichia coli. The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter. The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes. The lacUV5 promoter should be used when the wild-type expression level is low, the tacI promoter when the latter is high. The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lacYam mutation (eliminating lactose permease, the transport system for the inducer isopropyl-thio-beta-D-galactoside) is introduced. The method was demonstrated experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H+-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times the wild-type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level. We show that the introduction of a lac Y mutation facilitated the attainment of steady induction levels of partially induced cells. The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl-thio-beta-D-galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values. These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.
    Original languageEnglish
    JournalEuropean Journal of Biochemistry
    Volume211
    Issue number1-2
    Pages (from-to)181-191
    Number of pages11
    ISSN0014-2956
    DOIs
    Publication statusPublished - 1993

    Keywords

    • Cloning, Molecular
    • Escherichia coli
    • Escherichia coli Proteins
    • Gene Expression Regulation, Bacterial
    • Genes, Bacterial
    • Genetic Engineering
    • Lac Operon
    • Membrane Transport Proteins
    • Monosaccharide Transport Proteins
    • Operon
    • Plasmids
    • Promoter Regions, Genetic
    • Symporters
    • LacY protein, E coli
    • 9068-45-5 lactose permease
    • BIOCHEMISTRY
    • BOUND ATP-SYNTHASE
    • ESCHERICHIA-COLI
    • DNA
    • OVERPRODUCTION
    • REPRESSION
    • SEQUENCES
    • PATHWAY
    • GROWTH
    • FLUX
    • ENZYME
    • GENE EXPRESSION
    • Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli
    • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
    • 10064, Biochemistry studies - Proteins, peptides and amino acids
    • 10300, Replication, transcription, translation
    • 10808, Enzymes - Physiological studies
    • 31500, Genetics of bacteria and viruses
    • Biochemistry and Molecular Biophysics
    • Enzymology
    • Genetics
    • Molecular Genetics

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