Fig. 1

Overall scheme of the hybrid input circuit and toehold switch-based modulator. a A previously characterized hybrid input riboswitch circuit combined an off-type coenzyme B₁₂ riboswitch with transcriptional repressors to invert the output signal [21]. b ON/OFF modes of the hybrid input circuit. In the absence of a ligand, the transcriptional repressor is expressed by the off-type coenzyme B₁₂ riboswitch and represses the transcription of GFP (green fluorescence protein). In the presence of a ligand, expression of the transcriptional repressor is inhibited, resulting in transcription of GFP. c Scheme of the toehold switch-based modulator circuit. The riboswitch controlled the expression of transcriptional repressors; however, in this study, the transcriptional repressors regulated the expression of trigger RNAs, rather than reporter proteins. Then, the interaction between the toehold switch and trigger RNA activates the expression of the reporter gene. d ON/OFF modes of the toehold switch-based modulator circuit. In the absence of a ligand, the riboswitch and transcriptional repressor is transcribed by the constitutive promoter. Since the riboswitch is off type, the translation of the transcriptional repressor is allowed, which in turn represses the transcription of trigger RNA. Consequently, the toehold switch RNA maintains the translation-repressing hairpin structure, occluding the ribosomal binding site (RBS) of the reporter gene. On the contrary, in the presence of a ligand, expression of the transcriptional repressor is inhibited, resulting in robust transcription of trigger RNAs. The binding of the trigger RNA to the cognate toehold switch RNA exposes the RBS to activate translation of the reporter gene. The P_J23xxx, P_PhlF, P_T7 refer to the Anderson promoter series, PhlF transcriptional repressor cognate promoter, and T7 promoter, respectively