Fig. 5

Fluorescence distribution of single B. subtilis cells producing varying amounts of GUS11 analyzed by flow cytometry. Cultures of B. subtilis DB430 harboring plasmids pBS-Xnt-GUS11 and pHT01-iSplitGFP for expression of gus11 with varying spacers from 4 to 12 nucleotides (as indicated by Xnt) and the detector protein were grown at 30 °C. For the induction of detector gene expression cultures were supplemented with 1 mM IPTG prior to inoculation. As negative controls, both an empty vector control (EV) and a pBS-8nt-GUS-11 variant without induction of detector expression (NI) were included. Culture samples were collected at the late stationary growth phase (after 24 h) and analyzed by flow cytometry. The cells were gated based on their respective FSC and SSC signals to exclude cell debris and accumulation of cells (see Additional file 1: Figure S4). The iSplit GFP fluorescence intensity of each cell was measured and plotted against the frequency of the signal intensities. The percentages of fluorescent to non-fluorescent cells separated by a line are shown in each graph. All graphs are representative examples of triplicate measurements