Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity.

Abstract

Expression systems based on the Escherichia coli arabinose operon P(BAD) promoter exhibit the all-or-nothing (autocatalytic) induction of expression that was first documented in the lac operon. Under conditions of subsaturating levels of inducer, some of the cells of the population are fully induced, whereas other cells remain uninduced. Recently, a new AraE transporter system was reported to have circumvented the problem of autocatalytic expression in the pBAD expression vectors and to provide graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations [Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A. & Keasling, J. D. (2000) J. Bacteriol. 182, 7029-7034]. However, we report that nonuniform gene expression in the AraE system was readily detectable by the use of mutant green fluorescent proteins that are rapidly degraded in E. coli. We report an approach to avoid all-or-nothing induction of the pBAD promoter; the use of a mutant LacY transporter in a strain deficient in both arabinose transport (araE araFGH) and degradation (araBAD). This mutant LacY protein performs facilitated diffusion of arabinose resulting in homogeneous expression of an unstable GFP that is maintained over extended incubation times at subsaturating levels of inducer. This approach is readily adapted to other sugar-regulated expression systems.