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.

Overloaded enzymatic processes are shown to create indirect coupling between upstream components in cellular networks. This has important implications for the design of synthetic biology devices and for our understanding of currently inexplicable links within endogenous biological systems.

Robust oscillations have recently been observed in a synthetic gene network composed of coupled positive and negative feedback loops. Here we use deterministic and stochastic modeling to investigate how a small time delay in such regulatory networks can lead to strongly nonlinear oscillations that can be characterized by “degrade and fire” dynamics. We show that the period of the oscillations can be significantly greater than the delay time, provided the circuit components possess strong activation and tight repression. The variability of the period is strongly influenced by fluctuations near the oscillatory minima, when the number of regulatory molecules is small. We explore the effect of the positive feedback loop on the robustness of these oscillations.

Bacterial colonies often exhibit complex spatio-temporal organization. This collective behavior is affected by a multitude of factors ranging from the properties of individual cells (shape, motility, membrane structure) to chemotaxis and other means of cell–cell communication. One of the important but often overlooked mechanisms of spatio-temporal organization is direct mechanical contact among cells in dense colonies such as biofilms. While in natural habitats all these different mechanisms and factors act in concert, one can use laboratory cell cultures to study certain mechanisms in isolation. Recent work demonstrated that growth and ensuing expansion flow of rod-like bacteria Escherichia coli in confined environments leads to orientation of cells along the flow direction and thus to ordering of cells. However, the cell orientational ordering remained imperfect. In this paper we study one mechanism responsible for the persistence of disorder in growing cell populations. We demonstrate experimentally that a growing colony of nematically ordered cells is prone to the buckling instability. Our theoretical analysis and discrete-element simulations suggest that the nature of this instability is related to the anisotropy of the stress tensor in the ordered cell colony.

Flows of cells growing as a quasimonolayer in a confined space can exhibit streaming, with narrow streams of fast-moving cells flowing around clusters of slowly moving cells. We observed and analyzed this phenomenon experimentally for E. coli bacteria proliferating in a microfluidic cell trap using time-lapse microscopy. We also performed continuum modeling and discrete-element simulations to elucidate the mechanism behind the streaming instability. Our analysis demonstrates that streaming can be explained by the interplay between the slow adaptation of a cell to its local microenvironment and its mobility due to changes of cell-substrate contact forces.