Fig. 4. SAGE enables stable expression of heterologous genes in the absence of selection.

Flow cytometry measurements of fluorescence in populations of P. fluorescens JE4621 containing either no heterologous expression construct (parent strain), a pBBR-based replicating mNeonGreen expression vector (pJE354), or a nonreplicating mNeonGreen expression vector that has been chromosomally integrated at the indicated attB site by the corresponding recombinase. Starter cultures, with the exception of the parent strain that was grown in the absence of antibiotic, were cultivated in medium containing kanamycin sulfate (50 μg/ml) for plasmid selection. Each passage was inoculated with a 1024-fold dilution of its precursor culture (starter → passage 1 → passage 2) to enable 10 generations of exponential growth before reaching stationary phase. The x axis indicates the relative fluorescence (excitation at 488 nm and emission at 530 nm) of each cell. The y axis represents the abundance of cells in the population with a given relative fluorescence unit (RFU).