The use of lac-type promoters in control analysis.

Jensen PR, Westerhoff HV, Michelsen O

European journal of biochemistry

Jan 15, 1993

Sources

2 min read

0:00 / 0:00

Sign in to access this feature

Create a free account or sign in to use AI summaries, listen to articles, download PDFs, and save to your library.

1 view

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 lacY7am 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 lacY 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.