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A single‐cell assay of β‐galactosidase in recombinant Escherichia coli using flow cytometry

A flow cytometric method was developed for the assay of β‐galactosidase in single Escherichia coli cells. A new fluorogenic substrate for β‐galactosidase, C12FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular β‐galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained β‐galactosidase–positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid‐bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular β‐galactosidase along with a rapid increase in the fraction of plasmid‐free cells. Once the cells lose the plasmid, they no longer produce β‐galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid‐bearing cells can be distinguished from the nonfluorescent, plasmid‐free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between β‐galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction. © 1993 John Wiley & Sons, Inc.