Figure 1.

Schematics of the synthetic networks. Pointed and blunt arrows denote activation and repression, respectively. In all constructs, the output protein and the repressor are transcribed from the same bidirectional promoter in the constant and non-limiting presence of the TF rtTA that does not constitute an input to the system. In the diagrams, I indicates input node, I_R is the input reporter, A is the auxiliary regulator, and O is the output. (A) Transcriptional type I incoherent feedforward motif (tI1-FFL): the output protein is DsRed and the auxiliary repressor is LacI. The plasmid copy number is reported by the ZsGreen1 fluorescent protein, cotranslated with the LacI protein using IRES. Corresponding control circuit is also shown. (B) Post-transcriptional type I incoherent feedforward motif version I (ptI1-FFL_I): the output protein is AmCyan and the auxiliary repressor is a microRNA. Plasmid copy number is reported by the DsRed protein coexpressed with the output. DsRed mRNA also contains an intron coding for the regulator microRNA. Corresponding control circuit is also shown. (C) Post-transcriptional type I incoherent feedforward motif version II (ptI1-FFL_II): the output protein is DsRed, repressed by a microRNA processed from the intron in its own mRNA. The input is reported by the divergently expressed AmCyan protein. The control circuit is identical to the one in (B). (D) Transcriptional negative autoregulation motif (tAM): auxiliary repressor LacI becomes the output and represses its own transcription as well as the level of the ZsGreen output reporter contranslated via IRES. The input is reported by a divergently expressed DsRed protein.