Figure 3

Schematic diagram demonstrating the protocols for efflux assay and competitive growth enrichment. (a) Efflux assay was performed in the acrB-inactivated E. coli strain JA300A transformed with the plasmids of interest. Expressed AcrB were inactivated to allow substantial uptake of n- octane. Resuspension of the cells in assay buffer containing glucose reactivated the cells and the change in intracellular n- octane content was quantified by GC-MS to determine the efflux rate. Discrete clones were isolated from libraries with improved AcrB mutants. These mutants were assayed using the same method except the cells were cultivated from single colonies. (b) JA300A was transformed with plasmids harboring acrB mutants. The cultures containing cells expressing a mixture of AcrB variants were grown in the presence of n- octane or 1-octanol. As inferior mutants were killed by the hydrocarbons, the population became enriched with superior AcrB that conferred increased tolerance. The plasmid mixtures were isolated after enrichment for efflux assay.