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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.

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

We study both in silico and in vivo the real-time feedback control of a molecular titration motif that has been earmarked as a fundamental component of antithetic and multicellular feedback control schemes in E. coli. We show that an external feedback control strategy can successfully regulate the average fluorescence output of a bacterial cell population to a desired constant level in real-time. We also provide in silico evidence that the same strategy can be used to track a time-varying reference signal where the set-point is switched to a different value halfway through the experiment. We use the experimental data to refine and parameterize an in silico model of the motif that can be used as an error computation module in future embedded or multicellular control experiments.