Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture

The arabinose-inducible promoter P-BAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulati ons of cells that are fully induced and uninduced. To construct a host-vect or expression system with regulatable control in a homogeneous population o f cells, the araE gene of Escherichia coil was cloned into an RSF1010-deriv ed plasmid under control of the isopropyl-beta -D-thiogalactopyranoside-ind ucible P-tac and P-taclac promoters. This gene encodes the low-affinity, hi gh-capacity arabinose transport protein and is controlled natively by an ar abinose-inducible promoter. To detect the effect of arabinose-independent a raE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promo ter (P-BAD) on the pMB1-derived plasmid pBAD24. The transporter and reporte r plasmids were transformed into E, coli strains with native arabinose tran sport systems and strains deficient in one or both of the arabinose transpo rt systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE ara FGH mutant strains harboring the transporter and reporter plasmids were uni formly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concent ration, In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.