ABSTRACT: 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:Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in Drosophila larvae, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. We also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.
Project description:Although the structure of a genetically encoded regulatory circuit is an important determinant of its function, the relationship between circuit topology and the dynamical behaviors it can exhibit is not well understood. Here, we explore the range of behaviors available to the AC-DC circuit. This circuit consists of three genes connected as a combination of a toggle switch and a repressilator. Using dynamical systems theory, we show that the AC-DC circuit exhibits both oscillations and bistability within the same region of parameter space; this generates emergent behaviors not available to either the toggle switch or the repressilator alone. The AC-DC circuit can switch on oscillations via two distinct mechanisms, one of which induces coherence into ensembles of oscillators. In addition, we show that in the presence of noise, the AC-DC circuit can behave as an excitable system capable of spatial signal propagation or coherence resonance. Together, these results demonstrate how combinations of simple motifs can exhibit multiple complex behaviors.
Project description:Synthetic biology has enabled the creation of biological reconfigurable circuits, which perform multiple functions monopolizing a single biological machine; Such a system can switch between different behaviours in response to environmental cues. Previous work has demonstrated switchable dynamical behaviour employing reconfigurable logic gate genetic networks. Here we describe a computational framework for reconfigurable circuits in E.coli using combinations of logic gates, and also propose the biological implementation. The proposed system is an oscillator that can exhibit tunability of frequency and amplitude of oscillations. Further, the frequency of operation can be changed optogenetically. Insilico analysis revealed that two-component light systems, in response to light within a frequency range, can be used for modulating the frequency of the oscillator or stopping the oscillations altogether. Computational modelling reveals that mixing two colonies of E.coli oscillating at different frequencies generates spatial beat patterns. Further, we show that these oscillations more robustly respond to input perturbations compared to the base oscillator, to which the proposed oscillator is a modification. Compared to the base oscillator, the proposed system shows faster synchronization in a colony of cells for a larger region of the parameter space. Additionally, the proposed oscillator also exhibits lesser synchronization error in the transient period after input perturbations. This provides a strong basis for the construction of synthetic reconfigurable circuits in bacteria and other organisms, which can be scaled up to perform functions in the field of time dependent drug delivery with tunable dosages, and sets the stage for further development of circuits with synchronized population level behaviour.
Project description:Gardner2000 - genetic toggle switch in E.coli
The behaviour of the genetic toggle switch and the conditions for bistability has been studies using a synthetic, bistable gene circuit.
This model is described in the article:
Construction of a genetic toggle switch in Escherichia coli.
Gardner TS, Cantor CR, Collins JJ
Nature. 2000 Jan 20;403(6767):339-42.
It has been proposed' that gene-regulatory circuits with virtually any desired property can be constructed from networks of simple regulatory elements. These properties, which include multistability and oscillations, have been found in specialized gene circuits such as the bacteriophage lambda switch and the Cyanobacteria circadian oscillator. However, these behaviours have not been demonstrated in networks of non-specialized regulatory components. Here we present the construction of a genetic toggle switch-a synthetic, bistable gene-regulatory network-in Escherichia coli and provide a simple theory that predicts the conditions necessary for bistability. The toggle is constructed from any two repressible promoters arranged in a mutually inhibitory network. It is flipped between stable states using transient chemical or thermal induction and exhibits a nearly ideal switching threshold. As a practical device, the toggle switch forms a synthetic, addressable cellular memory unit and has implications for biotechnology, biocomputing and gene therapy.
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Project description:Periodic oscillations play a key role in cell physiology from the cell cycle to circadian clocks. The interplay of positive and negative feedback loops among genes and proteins is ubiquitous in these networks. Often, delays in a negative feedback loop and/or degradation rates are a crucial mechanism to obtain sustained oscillations. How does nature control delays and kinetic rates in feedback networks? Known mechanisms include proper selection of the number of steps composing a feedback loop and alteration of protease activity, respectively. Here, we show that a remarkably simple means to control both delays and effective kinetic rates is the employment of DNA binding sites. We illustrate this design principle on a widely studied activator-repressor clock motif, which is ubiquitous in natural systems. By suitably employing DNA target sites for the activator and/or the repressor, one can switch the clock "on" and "off" and precisely tune its period to a desired value. Our study reveals a design principle to engineer dynamic behavior in biomolecular networks, which may be largely exploited by natural systems and employed for the rational design of synthetic circuits.
Project description:Progress in unravelling the cellular and molecular basis of mammalian circadian regulation over the past decade has provided us with new avenues through which we can explore central nervous system disease. Deteriorations in measurable circadian output parameters, such as sleep/wake deficits and dysregulation of circulating hormone levels, are common features of most central nervous system disorders. At the core of the mammalian circadian system is a complex of molecular oscillations within the hypothalamic suprachiasmatic nucleus. These oscillations are modifiable by afferent signals from the environment, and integrated signals are subsequently conveyed to remote central neural circuits where specific output rhythms are regulated. Mutations in circadian genes in mice can disturb both molecular oscillations and measurable output rhythms. Moreover, systematic analysis of these mutants indicates that they can express an array of abnormal behavioural phenotypes that are intermediate signatures of central nervous system disorders. Furthermore, the response of these mutants to psychoactive drugs suggests that clock genes can modify a number of the brain's critical neurotransmitter systems. This evidence has led to promising investigations into clock gene polymorphisms in psychiatric disease. Preliminary indications favour the systematic investigation of the contribution of circadian genes to central nervous system disease.
Project description: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:General anesthesia (GA) is a reversible drug-induced state of altered arousal required for more than 60,000 surgical procedures each day in the United States alone. Sedation and unconsciousness under GA are associated with stereotyped electrophysiological oscillations that are thought to reflect profound disruptions of activity in neuronal circuits that mediate awareness and cognition. Computational models make specific predictions about the role of the cortex and thalamus in these oscillations. In this paper, we provide in vivo evidence in rats that alpha oscillations (10-15 Hz) induced by the commonly used anesthetic drug propofol are synchronized between the thalamus and the medial prefrontal cortex. We also show that at deep levels of unconsciousness where movement ceases, coherent thalamocortical delta oscillations (1-5 Hz) develop, distinct from concurrent slow oscillations (0.1-1 Hz). The structure of these oscillations in both cortex and thalamus closely parallel those observed in the human electroencephalogram during propofol-induced unconsciousness. During emergence from GA, this synchronized activity dissipates in a sequence different from that observed during loss of consciousness. A possible explanation is that recovery from anesthesia-induced unconsciousness follows a "boot-up" sequence actively driven by ascending arousal centers. The involvement of medial prefrontal cortex suggests that when these oscillations (alpha, delta, slow) are observed in humans, self-awareness and internal consciousness would be impaired if not abolished. These studies advance our understanding of anesthesia-induced unconsciousness and altered arousal and further establish principled neurophysiological markers of these states.
Project description:We show that logic computational circuits in gene regulatory networks arise from a fibration symmetry breaking in the network structure. From this idea we implement a constructive procedure that reveals a hierarchy of genetic circuits, ubiquitous across species, that are surprising analogues to the emblematic circuits of solid-state electronics: starting from the transistor and progressing to ring oscillators, current-mirror circuits to toggle switches and flip-flops. These canonical variants serve fundamental operations of synchronization and clocks (in their symmetric states) and memory storage (in their broken symmetry states). These conclusions introduce a theoretically principled strategy to search for computational building blocks in biological networks, and present a systematic route to design synthetic biological circuits.
Project description:Activity in neocortex is often characterized by synchronized oscillations of neurons and networks, resulting in the generation of a local field potential (LFP) and electroencephalogram. Do the neuronal networks of the cerebellum also generate synchronized oscillations and are they under the influence of those in the neocortex? Here we show that, in the absence of any overt external stimulus, the cerebellar cortex generates a slow oscillation that is correlated with that of the neocortex. Disruption of the neocortical slow oscillation abolishes the cerebellar slow oscillation, whereas blocking cerebellar activity has no overt effect on the neocortex. We provide evidence that the cerebellar slow oscillation results in part from the activation of granule, Golgi, and Purkinje neurons. In particular, we show that granule and Golgi cells discharge trains of single spikes, and Purkinje cells generate complex spikes, during the "up" state of the slow oscillation. Purkinje cell simple spiking is weakly related to the cerebellar and neocortical slow oscillation in a minority of cells. Our results indicate that the cerebellum generates rhythmic network activity that can be recorded as an LFP in the anesthetized animal, which is driven by synchronized oscillations of the neocortex. Furthermore, we show that correlations between neocortical and cerebellar LFPs persist in the awake animal, indicating that neocortical circuits modulate cerebellar neurons in a similar manner in natural behavioral states. Thus, the projection neurons of the neocortex collectively exert a driving and modulatory influence on cerebellar network activity.