Fig. 2. Characterization of the pneumococcal CRISPRlator.

a Expression of ext-sgRNA2 represses transcription of the mTurquoise2 reporter (blue) and ext-sgRNA3; ext-sgRNA3 represses mNeonGreen (green) and ext-sgRNA1, and ext-sgRNA1 represses mScarlet-I (red) and ext-sgRNA2, leading to oscillatory behavior (~symbol). b Schematic overview of the here designed and used microfluidics device. The chip, measured from the outer edge of the inputs/output, is 14.3 × 6.8 mm. Bacteria were injected via the top inlet and C + Y media was pumped through the middle inlet at a flow rate of 0.5 ml/h. The bottom left inlet was blocked. The single waste outlet is shown on the right. Scale bar = 1.43 mm. c Zoom in on a mother machine lane within the microfluidic device. Mother machine channels are 1.7 µm wide, 1.25 µm high and have 3.4 µm wide side channels of 250 nm height to increase nutrient/waste flow. The main feeding/flow channel is 20 µm high and 60 µm wide. d Mean expression of a total lineage of cells over time in one mother machine lane shows red-green-blue oscillation over time. e Circular lineage tree of the same population shown in d, where color intensity is the mean fluorescence intensity of each single cell in the lineage (left-right: mScarletI, mNeonGreen and mTurquoise2). The ancestor cell is positioned in the middle of each tree.