Building biological memory by linking positive feedback loops

Abstract

A common topology found in many bistable genetic systems is two interacting positive feedback loops. Here we explore how this relatively simple topology can allow bistability over a large range of cellular conditions. On the basis of theoretical arguments, we predict that nonlinear interactions between two positive feedback loops can produce an ultrasensitive response that increases the range of cellular conditions at which bistability is observed. This prediction was experimentally tested by constructing a synthetic genetic circuit in Escherichia coli containing two well-characterized positive feedback loops, linked in a coherent fashion. The concerted action of both positive feedback loops resulted in bistable behavior over a broad range of inducer concentrations; when either of the feedback loops was removed, the range of inducer concentrations at which the system exhibited bistability was decreased by an order of magnitude. Furthermore, bistability of the system could be tuned by altering growth conditions that regulate the contribution of one of the feedback loops. Our theoretical and experimental work shows how linked positive feedback loops may produce the robust bistable responses required in cellular networks that regulate development, the cell cycle, and many other cellular responses.

Fig. 1.

Design and function of the genetic toggle switch. (A) Basic circuit design for the genetic toggle switch. The LacI repressor was produced from the natural wild-type lacI gene. The activator module containing the glnG structural gene was located in the rbs region of the E. coli chromosome. The activator and repressor competed for control of the expression of the activator module promoter; when neither activator nor repressor proteins were present, a weak promoter (not depicted) allowed for transcription of the activator gene (12). The reporter consisted of a fusion of the activator-dependent glnK promoter with the lacZ gene, located in the trp region of the chromosome. For the system as shown with a single positive feedback loop, the lacY gene was next to lacZ, but contained a null mutation. (B) Graphic representation of the factors affecting the range of IPTG concentrations at which bistability is obtained. All of the plots depict rate of activator production or destruction vs. the concentration of activator. The destruction of activator is not regulated by activator and thus is likely to have a slope of 1, as depicted. Conversely, the production of activator is known to display high kinetic order and is depicted as the S-shaped curve. The role of IPTG is to shift this S-shaped curve to the right or the left, as depicted. Steady states occur when the two curves intersect, as indicated by small circles. The dotted circle in the Center plot depicts an unstable steady state. Factors controlling the shape of the S-shaped activator production curve, such as the steepness of this curve or its absolute height, control the range of bistability of the system.

Fig. 2.

Strong bistability was obtained by linking distinct positive feedback loops. (A) Genetic toggle switch with a single positive feedback loop. (Left) Schematic depiction of the genetic system in which the genetic toggle switch drives the expression of the lacZYA operon, but the lacY gene contains a null mutation. (Right) Result of bistability experiment, showing an ∼12-fold range of IPTG concentrations at which the system displayed bistability. Symbols: , naive (uninduced) culture; , preinduced culture. (B) Genetic system with a single positive feedback loop based on galactoside permease. (Left) Schematic depiction of the genetic system where the lacZ promoter was used to drive the expression of glnG and the phosphorylated form of glnG drives the expression of lacZYA. The lacY product, galactoside permease, provides positive feedback by facilitating the uptake of IPTG, which inactivates repressor. (Right) Result of bistability experiment, showing an ∼4-fold range of inducer concentrations at which the system displayed bistability. Symbols are as in A. (C) Genetic system with two positive feedback loops. (Left) Schematic depiction of the genetic system in which the genetic toggle switch drives the expression of the lacZYA operon, as in A except with a wild-type lacY gene. (Right) Result of bistability experiment showing that bistability was obtained over an ∼480-fold range of IPTG. For A–C, cells were grown in minimal medium with succinate as the carbon source and glutamine as the nitrogen source.

Fig. 3.

Bistability of the double-toggle switch was tunable by growth substrates causing catabolite repression. (A and B) The double-toggle switch strain depicted in Fig. 2C was examined in medium containing succinate and casein hydrolysate (A) and in medium containing glucose and casein hydrolysate (B). (C) The experiment is as in B, but the strain contained a null mutation in lacY and thus had only a single functioning positive feedback loop.