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.

Design principles for robust oscillatory behavior

“…On the contrary, negative autoregulation interactions are absent in robust topologies. Our result for positive autoregulation agrees with earlier studies which also demonstrated numerically (Tsai et al 2008) and experimentally Stricker et al 2008;Tigges et al 2009) the importance of positive autoregulation. In terms of having negative feedback loops in the network, at least one loop is required, with most robust topologies using at least two (Fig.…”

“…Not only can this shed light into which parameter configuration makes a topology oscillate, but also into how ''tunable'' a topology is, that is, how much the oscillatory response can be controlled by changing the parameters of the network. Tunability, as well as robustness, is a highly desirable feature in synthetic oscillators (Tsai et al 2008;Stricker et al 2008). We started our analysis by studying the distribution of the robustness among all three-component topologies.…”

“…4) or by inserting a third component in the positive branch of the feedback loop (Motifs 3, 4). Motif 1 is a well-known circuit, the ''amplified negative feedback oscillator'', where the key characteristic is to obtain significantly faster activator than repressor dynamics (Stricker et al 2008;Tigges et al 2009;Purcell et al 2010;Lomnitz and Savageau 2014). Motif 2, on the other hand, is less common in the literature, and it accounts more for not-so-robust topologies (median Oscillatory robustness is calculated as the fraction of parameter sets that yielded oscillatory responses parameters were randomly sampled 3 Â 10 5 times per topology.…”

Synthetic circuits, devices and modules

“…Tigges et al designed an oscillator to enable autonomous, self-sustained and tunable oscillatory gene expression in mammalian cells based on an auto-regulated transcription control circuit encoding a positive and a time-delayed negative feedback loop (Tigges et al, 2009). Using a modeled network architecture comprising linked positive and negative feedback loops, Stricker et al engineered a fast, robust and tunable genetic oscillator, whose robustness is achieved by introduction of a time delay in the negative feedback loop (Stricker et al, 2008). The synthetic oscillators provide insights into the dynamics of natural periodic processes and foster advances in the design of networks in future gene and cell therapies.…”

Motility versus fluctuations in mixtures of self-motile and passive agents

“…For example, techniques of synthetic biology and systems biology [8][9][10][11][12][13] have made it possible to fabricate bacterial strains with engineered gene-regulation circuits that produce predefined spatial and temporal patterns. Similarly, artificial self-motile agents can be realized through catalytically driven Janus particles [14][15][16][17][18].…”