Building biological memory by linking positive feedback loops.

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.

The robustness of the Escherichia coli signal-transducing UTase/UR-PII covalent modification cycle to variation in the PII concentration requires very strong inhibition of the UTase activity of UTase/UR by glutamine.

Uridylyltransferase/uridylyl-removing enzyme (UTase/UR) catalyzes uridylylation of PII and deuridylylation of PII-UMP, with both activities regulated by glutamine. In a reconstituted UTase/UR-PII cycle containing wild-type UTase/UR, the steady-state modification of PII varied from nearly complete modification to nearly complete demodification as glutamine was varied, whether the level of PII was saturating or unsaturating, but when a His-tagged version of UTase/UR was used, the robustness to variations in PII concentration was lost and the range of PII modification states in response to glutamine became smaller as the PII concentration increased. The presence of the His tag on UTase/UR did not alter PII substrate inhibition of the UT activity and had little effect on the level of the UT activity but resulted in a slight defect in UR activity. Importantly, at high PII concentrations, glutamine inhibition of the UT activity was incomplete. We hypothesized that binding of PII to the UR active site in the HD domain was responsible for PII substrate inhibition of the UT activity and, in the His-tagged enzyme, also weakened glutamine inhibition of the UT activity. Consistent with this, three different UTase/UR proteins with HD domain alterations lacked substrate inhibition of UT activity by PII; in one case, the HD alteration eliminated glutamine regulation of UT activity, while for the other two proteins, alterations of the HD domain partially compensated for the effect of the His tag in restoring glutamine regulation of UT activity. We conclude that very strong inhibition of UT activity was required for the UTase/UR-PII cycle to display robustness to the PII concentration, that in the wild-type enzyme PII brings about substrate inhibition of the UT activity by binding to the HD domain of the enzyme, and that addition of an N-terminal His tag resulted in an altered enzyme with subtle changes in the interactions between domains such that binding of PII to the HD domain interfered with glutamine regulation of the UT domain.

Using two-component systems and other bacterial regulatory factors for the fabrication of synthetic genetic devices.

Synthetic biology is an emerging field in which the procedures and methods of engineering are extended living organisms, with the long-term goal of producing novel cell types that aid human society. For example, engineered cell types may sense a particular environment and express gene products that serve as an indicator of that environment or affect a change in that environment. While we are still some way from producing cells with significant practical applications, the immediate goals of synthetic biology are to develop a quantitative understanding of genetic circuitry and its interactions with the environment and to develop modular genetic circuitry derived from standard, interoperable parts that can be introduced into cells and result in some desired input/output function. Using an engineering approach, the input/output function of each modular element is characterized independently, providing a toolkit of elements that can be linked in different ways to provide various circuit topologies. The principle of modularity, yet largely unproven for biological systems, suggests that modules will function appropriately based on their design characteristics when combined into larger synthetic genetic devices. This modularity concept is similar to that used to develop large computer programs, where independent software modules can be independently developed and later combined into the final program. This chapter begins by pointing out the potential usefulness of two-component signal transduction systems for synthetic biology applications and describes our use of the Escherichia coli NRI/NRII (NtrC/NtrB) two-component system for the construction of a synthetic genetic oscillator and toggle switch for E. coli. Procedures for conducting measurements of oscillatory behavior and toggle switch behavior of these synthetic genetic devices are described. It then presents a brief overview of device fabrication strategy and tactics and presents a useful vector system for the construction of synthetic genetic modules and positioning these modules onto the bacterial chromosome in defined locations.

Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli.

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.

Governor of the glnAp2 promoter of Escherichia coli.

Low-affinity sites for the activator NRI-P (NtrC-P) that map between the enhancer and the glnAp2 promoter were responsible for limiting promoter activity at high concentrations of NRI approximately P in intact cells and in an in vitro transcription system consisting of purified bacterial components. That is, the low-affinity sites constitute a 'governor', limiting the maximum promoter activity. As the governor sites are themselves far from the promoter, they apparently act either by preventing the formation of the activation DNA loop that brings the enhancer-bound activator and the promoter-bound polymerase into proximity or by preventing a productive interaction between the enhancer-bound activator and polymerase. The combination of potent enhancer and governor sites at the glnAp2 promoter provides for efficient activation of the promoter when the activator concentration is low, while limiting the maximum level of promoter activity when the activator concentration is high.

Activation of the glnA, glnK, and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation.

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI approximately P (NtrC approximately P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in Escherichia coli.

Analysis of the system design principles of signaling systems requires model systems where all components and regulatory interactions are known. Components of the Lac and Ntr systems were used to construct genetic circuits that display toggle switch or oscillatory behavior. Both devices contain an "activator module" consisting of a modified glnA promoter with lac operators, driving the expression of the activator, NRI. Since NRI activates the glnA promoter, this creates an autoactivated circuit repressible by LacI. The oscillator contains a "repressor module" consisting of the NRI-activated glnK promoter driving LacI expression. This circuitry produced synchronous damped oscillations in turbidostat cultures, with periods much longer than the cell cycle. For the toggle switch, LacI was provided constitutively; the level of active repressor was controlled by using a lacY mutant and varying the concentration of IPTG. This circuitry provided nearly discontinuous expression of activator.