Figure 1.

Synthetic circuits that perform diverse functions can be coupled to achieve higher-order responses. (a) Interlinked positive and negative feedback loops of different strengths drive an oscillatory response. Arabinose-responsive transcriptional activator (AraC) expression positively modulates gene expression and results in a positive feedback loop, whereas the isopropyl-β-D-thio-galactoside (IPTG)-responsive inhibitor of the lac promoter (LacI) inhibits expression and generates a negative feedback loop. The small-molecule inducers arabinose and IPTG modulate the strength of these feedback loops [42]. GFP, green fluorescent protein. (b) A mammalian AND gate composed of RNA interference (RNAi) target sites evaluates small interfering (si)RNA inputs. Unique RNAi target sites are placed in the 3' UTR of two lacI genes, and LacI regulates the expression of a fluorescent reporter, resulting in an AND logic evaluator for the siRNA inputs m1 and m2 [52]. YFP, yellow fluorescent protein. (c) Quorum-sensing circuitry allows population control. Cell density is broadcast by the diffusible small molecule acyl-homoserine lactone (AHL), which is synthesized by the enzyme LuxI (X). As cell density and AHL concentration increase, LuxR (R), a transcriptional regulator, binds AHL and initiates expression of a 'killer' gene (encoding CcdB, a lethal protein that targets the DNA gyrase complex), ultimately reducing the steady-state cell density [58]. (d) Interlinking positive and negative feedback loops with communication circuitry enables oscillation synchronization across a population of cells. Expression of R positively regulates expression of X, R, GFP, and AiiA (A), an enzyme that degrades AHL. As A increases in concentration, it degrades AHL and negatively modulates protein expression levels [62]. (e) Combining logic processing with communication circuitry enables a synthetic biological edge detection system. The expression of X and the transcriptional repressor cI (Y) is turned ON in cells in the dark region, where Y represses the expression of the pigment-producing protein (pigment: β-galactosidase, an enzyme that cleaves a substrate to produce a black pigment). However, diffusion of AHL synthesized by cells in the dark region activates R in cells at the edge of the light region (where Y is turned OFF), thus turning ON expression of pigment only in cells along this edge [63].