Supplementary Text A–C, Supplementary Figs A–F.

Text A, Image processing. Text B, Gene expression noise. Text C, Sender—receiver system. Fig A, Schematic overview of the bacterial sender-receiver system. Sender cells: As indicated, in the presence of IPTG repressor protein LacI is not bound to the lac promoters PLacUV5 on the bacterial genome and PT7lac on the sender plasmid. T7 RNA polymerase is then expressed, which in turn leads to the expression of AHL synthase LuxI and fluorescent reporter protein RFP from the plasmid. LuxI catalyzes the production of the quorum sensing signal N-3-oxo-C6-homoserine lactone (AHL), which can freely pass through the bacterial cell wall. Receiver cells constitutively express activator LuxR from the receiver plasmid. In the presence of AHL, LuxR activates GFP expression, which is under the control of the lux promoter Plux. In the first set of experiments in the main paper, only receiver cells are used and AHL is manually added to the culture medium to induce gene expression. Fig B, Microfluidic chemostats. (A) The microfluidic chemostat consists of a gradient mixer (adopted from Ref. 55 of the main paper), which generates linear concentration gradients of chemicals supplied through inlets 1 and 2, respectively. Eight gradient exits are connected to a total of 2 × 8 microfluidic channels, which contain trapping regions for bacteria (similar to Ref. 8 of the main paper). In the experiments, the concentration of AHL was varied in 1 nM steps in the range 0–21 nM, and in 10 nM steps in the range 20–90 nM. (B) Top view of a supply channel (blue) with trap region (grey). (C) Side view (not drawn to scale) showing the reduced height of the trap region, which only allows bacterial growth in a single layer. Fig C, Calibration of the gradient mixer system. We performed a series of calibraton experiments (with flow rates 40, 80, 160 and 320 μl/h) to evaluate the quality of the concentration gradient generated by the microfluidic mixer shown in Fig B in S1 File. In these experiments the right reservoir was loaded with buffer solution containing 10 μM fluorescein and the left reservoir with pure buffer (0 μM). After establishment of a steady gradient, we measured the fluorescence in the trap regions. The background-subtracted fluorescence values were then plotted against the nominal concentrations expected for the traps. As shown in the figure (which is obtained for the 160μl/h case), indeed a linear concentration gradient is generated. A linear regression fit to these values (fixed at 0μM) allows us to estimate the concentration errors. The maximum relative deviation from the nominal concentration is found to be ≈ 20% in all experiments performed. Fig D, Overview of the image analysis procedure. (A) A composite brightfield and fluorescence image, cropped to display only the microchamber contents. The program workflow is demonstrated by focusing on the red highlighted area of the picture, a region with dimensions 21.6×12.4 μm2. (B) Once the brightfield image is imported, contrast is enhanced and resolution increased. (C) Background detection is performed via a hybrid method combining adaptive thresholding and geometry information. (D) Cell markers are created using gradient information and geometric priors, refined using the watershed method. (E) A statistical classifier is used to remove mis-segmented cells. (F-I) Using the maximum overlap method, cell lineages are reconstructed (see also Fig D in S1 File). A cell division event is highlighted in (F-G); and propagated forward in (H-I). The user can correct tracking errors manually in the application. Fig E, Example of a cell lineage extracted using the segmentation software. The lineage is first automatically calculated by using the maximum overlap method on the segmented cells, as described in the main text. The segmentation method is conservative in detecting cell divisions, which means that already divided cells may be detected as a single cell for a few frames longer. This explains the observed cell division timings in the above lineage tree. After this step a correction heuristic is applied which finds potential mother-daughter mismatches by searching for fluorescence fluctuations twice as large as the calculated noise in a typical trajectory. For presentational clarity any branches which do not reach the final frame (due to mismatches) were manually edited out of the above plot. Fig F, Bulk analysis of gene induction by AHL using plate reader measurements. (A) Background subtracted absorbance of growing bacterial cultures for AHL concentrations ranging from 0 nM to 100 nM. (B) Corresponding background subtracted fluorescence intensities for the different AHL concentrations. (C) Maximum gene expression rate αmax obtained for the different AHL concentrations as explained in the main text. The solid line is a fit with a Hill curve with Hill exponent n = 0.97±0.08 and induction threshold K = 13.9±1.7 nM. (PDF)