The use of lac‐type promoters in control analysis

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