Gene networks and signalling pathways display complex topologies and, as a result, complex nonlinear behaviours. Accumulating evidence shows that both static (concentration) and dynamical (rate-of-change) features of transcription factors, ligands and environmental stimuli control downstream processes and ultimately cellular functions. Currently, however, methods to generate stimuli with desired features to probe cell response are still lacking. Here, combining tools from Control Engineering and Synthetic Biology (Cybergenetics), we propose a simple and cost-effective microfluidics-based platform to precisely regulate gene expression and signalling pathway activity in mammalian cells by means of real-time feedback control. We show that this platform allows: (i) to automatically regulate gene expression from inducible promoters in different cell types, including mouse embryonic stem cells; (ii) to precisely regulate the activity of the mTOR signalling pathway in single cells; (iii) to build a bio-hybrid oscillator in single embryonic stem cells by interfacing biological parts with virtual in silico counterparts. Ultimately, this platform can be used to probe gene networks and signalling pathways to understand how they process static and dynamic features of specific stimuli, as well as for the rapid prototyping of synthetic circuits for biotechnology and biomedical purposes.

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Summary Aggregation of α-synuclein and formation of inclusions are hallmarks of Parkinson’s disease (PD). Aggregate formation is affected by cellular environment, but it has been studied almost exclusively in cell-free systems. We quantitatively analyzed α-synuclein inclusion formation and clearance in a yeast cell model of PD expressing either wild-type (WT) α-synuclein or the disease-associated A53T mutant from the galactose (Gal)-inducible promoter. A computer-controlled microfluidics device regulated α-synuclein in cells by means of closed-loop feedback control. We demonstrated that inclusion formation is strictly concentration dependent and that the aggregation threshold of the A53T mutant is about half of the WT α-synuclein (56%). We chemically modulated the proteasomal and autophagic pathways and demonstrated that autophagy is the main determinant of A53T α-synuclein inclusions’ clearance. In addition to proposing a technology to overcome current limitations in dynamically regulating protein expression levels, our results contribute to the biology of PD and have relevance for therapeutic applications.

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified "hybrid" versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

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A major goal in synthetic development is to design and construct gene regulatory circuits that con- trol the patterning and morphogenesis of synthetic multicellular structures. In natural development, an interplay between mechanical and chemical communication shapes the dynamics of gene reg- ulatory circuits that underlie patterning and morphogenesis. However, for synthetic gene circuits, how the non-genetic properties of the growth environment impact circuit behavior remains poorly understood. Here, we describe an occurrence of mechano-chemical coupling in synthetic contact- dependent synNotch patterning circuits demonstrating that cell density modulates the transduction of signal between a sender and receiver cell. By exploiting density-dependent signaling, we con- struct multicellular signal propagation circuits with synNotch and control the patterning outcome both temporally and spatially via cell density gradients established in vitro via plating or small- molecule mediated modulation of proliferation. Our work demonstrates that synthetic gene circuits can be critically impacted by their context, providing an alternate means for programming multi- cellular circuit patterning outcomes.