Fig. 2

L7Ae-CS3-based cascade and two-state switch. a The cascade includes both protein–protein regulation and post-transcriptional regulation by siRNA. The L7Ae-CS3 transcript incorporates four siRNA-FF5 target sites in the 3’UTR. TEVp, which is co-expressed with EYFP, is downregulated by siRNA-FF4 via target sites in its 3’UTR. EBFP is expressed in absence of L7AeCS3 (Stage 0) and repressed in the presence of the RBP (Stage 1). TEVp cleaves L7Ae-CS3, rescuing EBFP expression (Stage 2). siRNA-FF5 (Input 1) downregulates L7Ae-CS3 in the absence of TEVp. siRNA-FF4 (Input 2) knock down of TEVp results in high L7Ae-CS3 levels and thus in EBFP repression. b Simplified logic circuit and corresponding flow cytometry data. The cascade implements the two-input logic “siRNA-FF5 OR (NOT siRNA-FF4)” (top). Bar charts of the EBFP and EYFP circuit outputs as additional stages of the cascade and the inputs are added to the experiment, starting from just the reporter (Stage 0), and then adding L7Ae-CS3 (Stage 1), input siRNA-FF5 (Input 1) or TEVp-2A-EYFP (Stage 2), and siRNA-FF4 (Input 2). Data represent geometric mean and standard deviation of means of EBFP and EYFP normalized by transfection marker mKate for n = 3 replicates. ru relative units. Significant changes determined by unpaired t-test are indicated with asterisks. *p-value < 0.05. c Schematics of the L7Ae-CS3-based switch. TEVp-2A-EYFP includes two K-turn motifs in the 5’UTR and four target sites for siRNA-FF4 in the 3’UTR. L7Ae-CS3 mRNA harbors four siRNA-FF5 target sites in the 3’UTR. To monitor L7Ae-CS3 activity, we included EBFP reporter with two K-turn motifs in the 5’UTR. siRNA-FF4 and siRNA-FF5 are used to set and reset the state of the switch. d Flow cytometry data representing network behavior. Data represent geometric mean and standard deviation of means of EBFP and EYFP normalized by transfection marker mKate for n = 3 replicates. ru relative units. e Representative fluorescent micrographs of the switch showing the state of the switch regulated by siRNA-FF4 and siRNA-FF5. Scale bars indicate 200 μm. Data collected 48 h post transfection