(A) LuxR can crosstalk with C12 to activate pLux. Top panel: schematic diagram of a synthetic gene circuit where a constitutive promoter (gray arrow) regulates LuxR (purple rectangle) expression. LuxR protein (purple bars), when dimerized and bound with C6 or C12, can activate pLux (purple arrow) to induce GFP (green rectangle) expression. The autoinducers, genes, and promoters are color coded so that naturally paired partners are in the same color. Bottom panel: dose response of the circuit when induced with C6 (gray) or C12 (black). (B) LasR can crosstalk with pLux when bound with C12. Top panel: schematic diagram of a circuit similar to that in (A), where a constitutive promoter regulates LasR (cyan rectangle) expression. LasR protein, when bound with C6 or C12, can activate pLux to induce GFP expression. Bottom panel: Dose response of this circuit when induced with C6 (gray) or C12 (black). Bar heights are averages of three independent flow cytometry measurements shown as mean ± SD. (C) Summary of crosstalk induction of all 16 different combinations, including inductions by both chemicals and corresponding synthase genes. The four combinations shown in (A) and (B) are highlighted with a gray background. Quantified results for other combinations are included in .

(A) Schematic diagram of a synthetic gene circuit where the pLux promoter regulates expression of LuxR, which in turn can bind with C6 or C12 to further activate pLux, forming a positive feedback loop (shown as simplified diagram). GFP under the regulation of pLux serves as the readout for LuxR levels. All components are color coded similarly as in . (B) The average of three replicate flow cytometry measurements is plotted as a square with error bars for each dose of C6 induction, where red indicates Initial ON cells while blue denotes Initial OFF cells. Solid lines represent results calculated from model fittings. The bistable region ranges from 0 to 10⁻⁹ M C6. Labels 1 and 2 indicate representative experiments within the region to be shown as histograms in (D). (C) Similar experiments as in (B) but with C12 inductions. The bistable region ranges from 10⁻⁸ to 10⁻⁶ M C12. Labels 3 and 4 indicate representative experiments within the bistable region to be shown as histograms in (D). (D) Histograms of flow cytometry measurements labeled in (B) and (C). One representative measurement from each point is shown. No bimodal distributions are observed.

(A) Schematic diagram of a synthetic LasR-pLux positive feedback circuit. GFP under the regulation of pLux serves as the readout for LuxR levels. All components are color coded similarly to . (B) The average of three replicate flow cytometry measurements is plotted as a square with error bars for each dose of C12 induction. Blue denotes Initial OFF cells, while green and red indicate the Initial ON cells induced with 10⁻⁴ M C12 and 10⁻⁹ M C12 before being re-diluted into concentrations of C12, respectively. Labels 1, 2, 3, and 4 indicate experiments to be shown in detail as histograms in (C). (C) Histograms of flow cytometry measurements labeled in (B). One representative measurement from each point is shown. A bimodal distribution is only observed for label 3: which is Initial ON cells (induced with 10⁻⁹ M C12 before redilution) at 10⁻⁸ M C12. (D) DNA analysis for the Initial ON samples shown as red in (B). Top: Plasmid DNA was extracted and digested with EcoRI and PstI, and argarose gel electrophoresis results indicated gene mutation happened in samples with 10⁻⁸ M and higher doses of C12. Lane 1 is the wild-type plasmid as the control, lanes 2 to 9 are samples in 10⁻¹¹ to 10⁻⁴ M C12, and Lane 10 is the 1kb DNA marker. V: vector; F: wild-type DNA fragment (the LasR-pLux positive feedback circuit); M: mutated fragment. Bottom: Schematic representation of the mutation and the features of IS10 transposase insertion: the target site (first CGCGTAGCG) in the LasR gene, its duplication (second CGCGTAGCG) due to insertion of IS10 transposase, and the IS10 sequence (red box and shown in italics).

(A) Model predictions of GFP expression at several transposition rates: high (red, k3=3.6e-6), low (green, k3=4e-7), and none (blue, k3=0). Histograms were constructed from 8000 single cell stochastic simulations at 1000 (k3=3.6e-6) and 1900 (k3=0 and k3=4e-7) minutes. (B) Experimental validation of the model predictions in (A). Red and green curves correspond to the high and low transposition rates from (A), and they exhibit similar bi- and trimodal responses, respectively. No blue curve is included because mutation could not be eliminated entirely experimentally. (C) Representative stochastic simulations of single cell fluorescence starting from the ON state. All possible transitions are shown. Inset diagram illustrates all possible state transitions in the simulation. (D) Model predictions of GFP expression with low transposition rate showing temporal evolution of the population from primarily ON cells at an early time (green), to trimodal distributions at intermediate time (blue), eventually falling into a primarily Mutated state at late time (red). (E) Flow cytometry measurements taken at 12 hours (green), 24 hours (blue), and 32 hours (green). Populations show similar dynamics to those predicted by the model in (D), starting with a large ON peak, transitioning to a trimodal distribution, then into primarily Mutated or OFF cells.

(A) GFP fluorescence (top) and phase contrast (bottom) images of cells growing in the microfluidic chamber at 0, 8, 16, and 24 hours. Magnification: 40×. (B) Normalized fluorescence expression of representative cells from (A), showing similar behavior to that predicted by the model from . Four cells are colored corresponding to the scenarios in , and the other 11 cells are grey. Each trajectory follows one cell, with the trajectory branching as the cells divide. One frame equals five minutes. (C) Diagram of the mechanism for trimodality. Each “valley” represents one state. The blue curve represents the landscape at 37 °C, and the dotted grey curve is the landscape at 34 °C. At 37 °C, ON state cells can more easily transition to the Mutated state because of the low barrier; while at 34 °C, the barrier between ON and Mutated states increases, resulting in more ON cells transitioning to OFF state and promoting the emergence of trimodality.