A fast, robust and tunable synthetic gene oscillator.
ABSTRACT: One defining goal of synthetic biology is the development of engineering-based approaches that enable the construction of gene-regulatory networks according to 'design specifications' generated from computational modelling. This approach provides a systematic framework for exploring how a given regulatory network generates a particular phenotypic behaviour. Several fundamental gene circuits have been developed using this approach, including toggle switches and oscillators, and these have been applied in new contexts such as triggered biofilm development and cellular population control. Here we describe an engineered genetic oscillator in Escherichia coli that is fast, robust and persistent, with tunable oscillatory periods as fast as 13 min. The oscillator was designed using a previously modelled network architecture comprising linked positive and negative feedback loops. Using a microfluidic platform tailored for single-cell microscopy, we precisely control environmental conditions and monitor oscillations in individual cells through multiple cycles. Experiments reveal remarkable robustness and persistence of oscillations in the designed circuit; almost every cell exhibited large-amplitude fluorescence oscillations throughout observation runs. The oscillatory period can be tuned by altering inducer levels, temperature and the media source. Computational modelling demonstrates that the key design principle for constructing a robust oscillator is a time delay in the negative feedback loop, which can mechanistically arise from the cascade of cellular processes involved in forming a functional transcription factor. The positive feedback loop increases the robustness of the oscillations and allows for greater tunability. Examination of our refined model suggested the existence of a simplified oscillator design without positive feedback, and we construct an oscillator strain confirming this computational prediction.
Project description:Oscillations occur in a wide variety of cellular processes, for example in calcium and p53 signaling responses, in metabolic pathways or within gene-regulatory networks, e.g. the circadian system. Since it is of central importance to understand the influence of perturbations on the dynamics of these systems a number of experimental and theoretical studies have examined their robustness. The period of circadian oscillations has been found to be very robust and to provide reliable timing. For intracellular calcium oscillations the period has been shown to be very sensitive and to allow for frequency-encoded signaling. We here apply a comprehensive computational approach to study the robustness of period and amplitude of oscillatory systems. We employ different prototype oscillator models and a large number of parameter sets obtained by random sampling. This framework is used to examine the effect of three design principles on the sensitivities towards perturbations of the kinetic parameters. We find that a prototype oscillator with negative feedback has lower period sensitivities than a prototype oscillator relying on positive feedback, but on average higher amplitude sensitivities. For both oscillator types, the use of Michaelis-Menten instead of mass action kinetics in all degradation and conversion reactions leads to an increase in period as well as amplitude sensitivities. We observe moderate changes in sensitivities if replacing mass conversion reactions by purely regulatory reactions. These insights are validated for a set of established models of various cellular rhythms. Overall, our work highlights the importance of reaction kinetics and feedback type for the variability of period and amplitude and therefore for the establishment of predictive models.
Project description:Many organisms have evolved molecular clocks to anticipate daily changes in their environment. The molecular mechanisms by which the circadian clock network produces sustained cycles have extensively been studied and transcriptional-translational feedback loops are common structures to many organisms. Although a simple or single feedback loop is sufficient for sustained oscillations, circadian clocks implement multiple, complicated feedback loops. In general, different types of feedback loops are suggested to affect the robustness and entrainment of circadian rhythms. To reveal the mechanism by which such a complex feedback system evolves, we quantify the robustness and light entrainment of four competing models: the single, semi-dual, dual, and redundant feedback models. To extract the global properties of those models, all plausible kinetic parameter sets that generate circadian oscillations are searched to characterize their oscillatory features. To efficiently perform such analyses, we used the two-phase search (TPS) method as a fast and non-biased search method and quasi-multiparameter sensitivity (QMPS) as a fast and exact measure of robustness to uncertainty of all kinetic parameters.So far the redundant feedback model has been regarded as the most robust oscillator, but our extensive analysis corrects or overcomes this hypothesis. The dual feedback model, which is employed in biology, provides the most robust oscillator to multiple parameter perturbations within a cell and most readily entrains to a wide range of light-dark cycles. The kinetic symmetry between the dual loops and their coupling via a protein complex are found critically responsible for robust and entrainable oscillations. We first demonstrate how the dual feedback architecture with kinetic symmetry evolves out of many competing feedback systems.
Project description:Biological systems exhibit numerous oscillatory behaviors from calcium oscillations to circadian rhythms that recur daily. These autonomous oscillators contain complex feedbacks with nonlinear dynamics that enable spontaneous oscillations. The detailed nonlinear dynamics of such systems remains largely unknown. In this paper, we investigate robustness and dynamical differences of five minimal systems that may underlie fundamental molecular processes in biological oscillatory systems. Bifurcation analyses of these five models demonstrate an increase of oscillatory domains with a positive feedback mechanism that incorporates a reversible reaction, and dramatic changes in dynamics with small modifications in the wiring. Furthermore, our parameter sensitivity analysis and stochastic simulations reveal different rankings of hierarchy of period robustness that are determined by the number of sensitive parameters or network topology. In addition, systems with autocatalytic positive feedback loop are shown to be more robust than those with positive feedback via inhibitory degradation regardless of noise type. We demonstrate that robustness has to be comprehensively assessed with both parameter sensitivity analysis and stochastic simulations.
Project description:Oscillations are an important feature of cellular signaling that result from complex combinations of positive- and negative-feedback loops. The encoding and decoding mechanisms of oscillations based on amplitude and frequency have been extensively discussed in the literature in the context of intercellular and intracellular signaling. However, the fundamental questions of whether and how oscillatory signals offer any competitive advantages-and, if so, what-have not been fully answered. We investigated established oscillatory mechanisms and designed a study to analyze the oscillatory characteristics of signaling molecules and system output in an effort to answer these questions. Two classic oscillators, Goodwin and PER, were selected as the model systems, and corresponding no-feedback models were created for each oscillator to discover the advantage of oscillating signals. Through simulating the original oscillators and the matching no-feedback models, we show that oscillating systems have the capability to achieve better resource-to-output efficiency, and we identify oscillatory characteristics that lead to improved efficiency.
Project description:The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells.
Project description:The engineering of genetic circuits with predictive functionality in living cells represents a defining focus of the expanding field of synthetic biology. This focus was elegantly set in motion a decade ago with the design and construction of a genetic toggle switch and an oscillator, with subsequent highlights that have included circuits capable of pattern generation, noise shaping, edge detection and event counting. Here we describe an engineered gene network with global intercellular coupling that is capable of generating synchronized oscillations in a growing population of cells. Using microfluidic devices tailored for cellular populations at differing length scales, we investigate the collective synchronization properties along with spatiotemporal waves occurring at millimetre scales. We use computational modelling to describe quantitatively the observed dependence of the period and amplitude of the bulk oscillations on the flow rate. The synchronized genetic clock sets the stage for the use of microbes in the creation of a macroscopic biosensor with an oscillatory output. Furthermore, it provides a specific model system for the generation of a mechanistic description of emergent coordinated behaviour at the colony level.
Project description:Nearly all living systems feature a temperature-independent oscillation period in circadian clocks. This ubiquitous property occurs at the system level and is rooted in the network architecture of the clock machinery. To investigate the mechanism of this prominent property of the circadian clock and provide general guidance for generating robust genetic oscillators with temperature-compensated oscillations, we theoretically explored the design principle and core network topologies preferred by oscillations with a temperature-independent period. By enumerating all topologies of genetic regulatory circuits with three genes, we obtained four network motifs, namely, a delayed negative feedback oscillator, repressilator, activator-inhibitor oscillator and substrate-depletion oscillator; hybrids of these motifs constitute the vast majority of target network topologies. These motifs are biased in their capacities for achieving oscillations and the temperature sensitivity of the period. The delayed negative feedback oscillator and repressilator are more robust for oscillations, whereas the activator-inhibitor and substrate-depletion oscillators are superior for maintaining a temperature-independent oscillation period. These results suggest that thermally robust oscillation can be more plausibly achieved by hybridizing these two categories of network motifs. Antagonistic balance and temperature insulation mechanisms for achieving temperature compensation are typically found in these topologies with temperature robustness. In the temperature insulation approach, the oscillation period relies on very few parameters, and these parameters are influenced only slightly by temperature. This approach prevents the temperature from affecting the oscillation period and generates circadian rhythms that are robust against environmental perturbations.
Project description:The ultimate goal of synthetic biology is the conception and construction of genetic circuits that are reliable with respect to their designed function (e.g. oscillators, switches). This task remains still to be attained due to the inherent synergy of the biological building blocks and to an insufficient feedback between experiments and mathematical models. Nevertheless, the progress in these directions has been substantial.It has been emphasized in the literature that the architecture of a genetic oscillator must include positive (activating) and negative (inhibiting) genetic interactions in order to yield robust oscillations. Our results point out that the oscillatory capacity is not only affected by the interaction polarity but by how it is implemented at promoter level. For a chosen oscillator architecture, we show by means of numerical simulations that the existence or lack of competition between activator and inhibitor at promoter level affects the probability of producing oscillations and also leaves characteristic fingerprints on the associated period/amplitude features.In comparison with non-competitive binding at promoters, competition drastically reduces the region of the parameters space characterized by oscillatory solutions. Moreover, while competition leads to pulse-like oscillations with long-tail distribution in period and amplitude for various parameters or noisy conditions, the non-competitive scenario shows a characteristic frequency and confined amplitude values. Our study also situates the competition mechanism in the context of existing genetic oscillators, with emphasis on the Atkinson oscillator.
Project description:Oscillatory dynamics are common in biological pathways, emerging from the coupling of positive and negative feedback loops. Due to the small numbers of molecules typically contained in cellular volumes, stochastic effects may play an important role in system behavior. Thus, for moderate noise strengths, stochasticity has been shown to enhance signal-to-noise ratios or even induce oscillations in a class of phenomena referred to as "stochastic resonance" and "coherence resonance," respectively. Furthermore, the biological oscillators are subject to influences from the division cycle of the cell. In this paper we consider a biologically relevant oscillator and investigate the effect of intrinsic noise as well as division cycle which encompasses the processes of growth, DNA duplication, and cell division. We first construct a minimal reaction network which can oscillate in the presence of large or negligible timescale separation. We then derive corresponding deterministic and stochastic models and compare their dynamical behaviors with respect to (i) the extent of the parameter space where each model can exhibit oscillatory behavior and (ii) the oscillation characteristics, namely, the amplitude and the period. We further incorporate division cycle effects on both models and investigate the effect of growth rate on system behavior. Our results show that in the presence but not in the absence of large timescale separation, coherence resonance effects result in extending the oscillatory region and lowering the period for the stochastic model. When the division cycle is taken into account, the oscillatory region of the deterministic model is shown to extend or shrink for moderate or high growth rates, respectively. Further, under the influence of the division cycle, the stochastic model can oscillate for parameter sets for which the deterministic model does not. The division cycle is also found to be able to resonate with the oscillator, thereby enhancing oscillation robustness. The results of this study can give valuable insight into the complex interplay between oscillatory intracellular dynamics and various noise sources, stemming from gene expression, cell growth, and division.
Project description:BACKGROUND: Feedback loops, both positive and negative are embedded in the Mitogen Activated Protein Kinase (MAPK) cascade. In the three layer MAPK cascade, both feedback loops originate from the terminal layer and their sites of action are either of the two upstream layers. Recent studies have shown that the cascade uses coupled positive and negative feedback loops in generating oscillations. Two plausible designs of coupled positive and negative feedback loops can be elucidated from the literature; in one design the positive feedback precedes the negative feedback in the direction of signal flow and vice-versa in another. But it remains unexplored how the two designs contribute towards triggering oscillations in MAPK cascade. Thus it is also not known how amplitude, frequency, robustness or nature (analogous/digital) of the oscillations would be shaped by these two designs. RESULTS: We built two models of MAPK cascade that exhibited oscillations as function of two underlying designs of coupled positive and negative feedback loops. Frequency, amplitude and nature (digital/analogous) of oscillations were found to be differentially determined by each design. It was observed that the positive feedback emerging from an oscillating MAPK cascade and functional in an external signal processing module can trigger oscillations in the target module, provided that the target module satisfy certain parametric requirements. The augmentation of the two models was done to incorporate the nuclear-cytoplasmic shuttling of cascade components followed by induction of a nuclear phosphatase. It revealed that the fate of oscillations in the MAPK cascade is governed by the feedback designs. Oscillations were unaffected due to nuclear compartmentalization owing to one design but were completely abolished in the other case. CONCLUSION: The MAPK cascade can utilize two distinct designs of coupled positive and negative feedback loops to trigger oscillations. The amplitude, frequency and robustness of the oscillations in presence or absence of nuclear compartmentalization were differentially determined by two designs of coupled positive and negative feedback loops. A positive feedback from an oscillating MAPK cascade was shown to induce oscillations in an external signal processing module, uncovering a novel regulatory aspect of MAPK signal processing.