FIGURE 1 

Design of synthetic switches and circuits. (A) Design of DNA sequences and reaction mechanisms. Each sequence subdomain is color-coded with its length in nucleotides written above it. For example, 62 base long inhibitor I₂ consists of five parts: the spacer sequence including the 6 base transcription initiation sequence (11 bases, green), the toehold-binding sequence (8 bases, light blue), the branch migration sequence (22 bases, red), the 5' end of the promoter sequence (5 bases, blue) and the 3' end hairpin structure (16 bases, brown). Switch templates have two distinct states, ON or OFF, with different transcription speed. The ON-state switch template (TA complex) has a complete promoter sequence (17 bases, blue) with a nick, while the OFF-state switch template (T) is missing 5 bases of the promoter sequence on the template side. The two single strands that form the switch templates are the longer nontemplate side strand, T-nt, and the shorter template side strand, T-t (Materials and methods). The T-nt strands are labeled with fluorophores (pink circle, Texas Red; yellow circle, TAMRA) and A strands are labeled with quenchers (black circles). Thus, the state of each switch can be monitored by measuring the fluorescence quenching efficiency (Marras et al, 2002). The ON-state source template has a complete promoter sequence with a nick (like the ON-state switch) and the OFF-state source template is missing five bases of the promoter sequence on the template side (like the OFF-state switch). Unlike the switch templates, source templates do not interact with activators, due to the hairpin stem permanently covering the branch migration sequence, and therefore maintain their transcription efficiency in the presence of inhibitors. The source activity can be controlled by preparing a mixture of ON and OFF source templates. The two single strands that form the source templates are the longer nontemplate side strand, So-nt (ON or OFF), and the shorter template side strand, T-t; the template side strands are the same for the switch and source templates that encode the same outputs (Materials and methods). For any given transcriptional circuit, we use either source template So_j (with some desired mixture of ON and OFF templates) or switch template Sw_ji to produce RNA inhibitor I_j. RNAP produces RNA inhibitors from DNA templates, while RNase H degrades RNA inhibitors bound to DNA activators. Detailed reaction mechanisms are listed in Box 1. (B) Two feedforward circuits where a source So_j controls a switch Sw_ij by supplying inhibitor I_j and a bistable system where two switches, Sw₁₂ and Sw₂₁, inhibit each other.