Fig. 3. Tunable formation of tree ring-like colonies.

a Three-day tree ring-like colony formation by thermally controlled expression of sfGFP and mRFP. Specifically, plates spread with recombinant cells from cell cultures at 37 °C were incubated in periodical changing circumstances at 37 °C and 30 °C for 10 h and 14 h, respectively. b The colony growth image was recorded every 24 h to study the forming process of rings in red or green. The band width of outer rings became more and more narrow due to the gradually saturated growth of colony, especially for green rings compared to that of the red ones. c Growth of a single colony harboring various T-switch circuits, including 155+165 from part b, 147+167 and 147+168 groups, by periodically changing temperature between 30 °C and 37 °C. Construct of 168 was derived from 167 by adding AAV degradation tag to reporter mrfp to reduce the memory effects of thermal controlled system. Fluorescence intensity (FI) was measured by imageJ from images and normalized by the maximum value of sfGFP and mRFP measured from every single image, ranging from 0 to 100. d Diameter of tree ring-like colonies increased with longer temperature cycles at 30 °C. e Ring-width of sfGFP- and mRFP-ring were obviously affected by different T-switch designs and temperature cycles from 30 °C to 37 °C. Hollow and solid squares represent the visible and missing formation of the first red ring (R1) generated by constructs 147+167, respectively, namely 147+167-D (2 colonies) and 147+167-S (4 colonies), respectively. Data in d and e are presented as mean ± s.d. of at least 2 collected colonies. p values of d and e are displayed in Supplementary Fig. 12.