Heatmap plots created using the R script in Figure 2.

<p>(<b>A</b>) <i>B. subtilis</i> cells expressing a fusion of the IPTG-inducible P<i>spac</i> promoter with GFP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068696#pone.0068696-Botella1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068696#pone.0068696-Buescher1" target="_blank">[7]</a> show homogeneous fluorescence. (<b>B</b>) <i>B. subtilis</i> cells expressing a fusion of the authentic promoter of the <i>sunA</i> gene to GFP show heterogeneous fluorescence when grown on a Luria Bertani agarose medium. (<b>C</b>) <i>B. subtilis</i> cells expressing the same <i>sunA</i> promoter GFP fusion as in B show bistable heterogeneous fluorescence when grown on an M9 agarose medium. Note that at early time points already two populations of cells with differing fluorescence intensities can be distinguished. AU, arbitrary units. (<b>D</b>) Bar diagrams for easy comparison of the outcomes of multiple heterogeneity measurements during growth on M9 medium as shown in panels A-C. At t = 2 h, cells are in the exponential growth phase (blue bars); at t = 5 h, the highest numbers of cells are observed (red bars); at t = 10 h, a minimum in the cell numbers has been reached due to cell death (green bars); and at t = 17 h, the surviving cells have resumed growth (purple bars). Heterogeneity is expressed in arbitrary units (AU).</p

Script used in the R software package to generate heatmap plots from ImageJ output data.

<p>Note that the indicated directories are arbitrary examples. The colors.csv file used in this script can be altered for implementing other color schemes.</p

Processing phase contrast images to create segmented cells.

<p>(A) ImageJ commands for the processing of phase contrast images to create segmented cells. (B) Visualization of the image processing from the original phase contrast image, through background subtraction, convolution, setting of a threshold grey intensity, conversion of values within threshold to mask and de-speckling. Red objects in the processed image are above the threshold and counted as cells. Notably, non-separated pairs of cells as marked with the white arrow pointing at the site of their attachment will be counted as one cell.</p

Quantification of expression heterogeneity.

<p>To obtain baseline values for GFP expression heterogeneity, a <i>B. subtilis</i> P<i>spac</i>-GFP strain was used in which the fluorescence intensity of the cells can be varied by varying the amount of the inducer IPTG in the growth medium. Importantly, the variation in GFP fluorescence in this cell population is minimal compared to cells expressing GFP from non-engineered promoters. Therefore, the observed variation can be regarded as a baseline for GFP expression heterogeneity. (<b>A</b>) Standard deviation in the fluorescence intensity of individual cells of <i>B. subtilis</i> P<i>spac</i>-GFP as a function of the mean fluorescence intensity of the cell population. The analysis included 535 measurements collected from four cultures supplemented with IPTG to 0.05 mM, 0.1 mM, 0.5 mM, or 1 mM. (<b>B</b>) Example to illustrate the effectiveness of the applied heterogeneity correction. When applied to a P<i>spac</i>-GFP strain grown in the presence of 0.05 mM IPTG the correction shows close to zero levels of expression heterogeneity over a period of at least 12 h. At later time points cells started to lyse in this experiment resulting in a slightly increased GFP expression heterogeneity. Black line, raw fluorescence data; Grey line, P<i>spac</i>-GFP subtracted data.</p