Project description:Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings.
Project description:Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives.
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Project description:Synthetic gene oscillators are small, engineered genetic circuits that produce periodic variations in target protein expression. Like other gene circuits, synthetic gene oscillators are noisy and exhibit fluctuations in amplitude and period. Understanding the origins of such variability is key to building predictive models that can guide the rational design of synthetic circuits. Here, we developed a method for determining the impact of different sources of noise in genetic oscillators by measuring the variability in oscillation amplitude and correlations between sister cells. We first used a combination of microfluidic devices and time-lapse fluorescence microscopy to track oscillations in cell lineages across many generations. We found that oscillation amplitude exhibited high cell-to-cell variability, while sister cells remained strongly correlated for many minutes after cell division. To understand how such variability arises, we constructed a computational model that identified the impact of various noise sources across the lineage of an initial cell. When each source of noise was appropriately tuned the model reproduced the experimentally observed amplitude variability and correlations, and accurately predicted outcomes under novel experimental conditions. Our combination of computational modeling and time-lapse data analysis provides a general way to examine the sources of variability in dynamic gene circuits.