(A) Schematic illustration.
(B) Reducing circuit-mediated metabolic burden, by reducing circuit copy number and on-demand circuit activation.
(C) Targeted killing of mutants. (Top) The toxin-antitoxin system induces cell death in the plasmid-free subpopulation that emerges due to plasmid segregation error. Toxin-producing host strain can survive only if carrying the plasmid encoding antitoxin. Once the cell loses the plasmid, toxin kills the host. (Bottom) A paradoxical circuit design, in which the same signal induces both cell growth and death, results in a biphasic effect of overall growth that depends on the signal intensity. In low-signal environments, cells with mutations that reduce their sensitivity to this signal are inherently selected against in this system, disfavoring the survival of such mutants.
(D) Direct coupling of circuit activation to survival. (Top) A gene of interest (GOI) and a survival gene is entangled by using an overlapping sequence; disruption of the GOI also disrupts the survival gene. (Bottom) Expression of a survival gene depends on the expression of the GOI; disruption of the latter would eliminate expression of the survival gene.