Figure 1.

Engineering genetic circuits at the single-cell level.

(A) A synthetic IMPLIES gate that is interfaced with an Inverter. As shown in the upper panel, the IMPLIES gate involves a constitutively expressed LacI repressor, which inhibits the transcription of CI from the P_lacI promoter; IPTG activates the P_lacI promoter and induces CI and ECFP expression; CI inhibits the λPRO12 promoter to repress EYFP expression. As shown in the bottom panel, the P_lacI promoter comprises an IMPLIES logic gate wherein two inputs, LacI and IPTG, induce the response of the output CI, which acts as the input to the interfaced Inverter based on the λPRO12 promoter. Arrows indicate synthesis. Blunt bars indicate repression. Figure adapted from Yokobayashi et al. [7].

(B) A synthetic riboregulated transcriptional cascade (RTC) counter circuit that can count two pulses of arabinose stimulations. The RTC two-counter includes a transcriptional cascade: promoter P_Ltet0-1 drives the transcription of T7 RNA polymerase (RNAP), whose protein binds the P_T7 promoter and mediates transcription of a green fluorescent protein (GFP). The two genes (T7 RNAP and GFP) are in turn repressed by the riboregulator (cr). Meanwhile, a short, transactivating noncoding RNA (taRNA), driven by the arabinose promoter P_BAD, can bind to cr, relieving its repression on RBS and effectively activating the translation of T7 RNAP and GFP proteins. Figure adapted from Friedland et al. [17].

(C) A tunable band-pass filter conferred by a two-arm feed-forward loop (FFL). In one arm, insufficient β-lactamase (BLA) activity causes accumulation of Amp, leading to compromised cell wall synthesis and repressed cell growth. In the second arm, cell wall (murein) breakdown of Amp results in the accumulation of aM-pentapeptide (aM-Pp), which activates the promoter P_ampC via interacting with the promoter's repressor AmpR. As a result, aM-Pp induces TetC synthesis (which confers cells' Tet resistance) and GFP expression. Alternatively, BLA can be expressed by IPTG induction of P_{tac P/O} promoter, which is repressed by LacI. Figure adapted from Sohka et al. [18].