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Applications of control engineering to mammalian cell biology have been recently implemented for precise regulation of gene expression. In this chapter, we report the main experimental and computational methodologies to implement automatic feedback control of gene expression in mammalian cells using a microfluidics/microscopy platform.

Advances in microscopy, microfluidics and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah – a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterise and control cells over time. We demonstrate Cheetah’s core capabilities by analysing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetah’s segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.

Applications of control engineering to mammalian cell biology have been recently implemented for precise regulation of gene expression. In this chapter, we report the main experimental and computational methodologies to implement automatic feedback control of gene expression in mammalian cells using a microfluidics/microscopy platform.

Extracting quantitative measurements from time-lapse images is necessary in external feedback control applications, where segmentation results are used to inform control algorithms. While such image segmentation applications have been previously reported, there is in the literature a lack of open-source and documented code for the community. We describe ChipSeg, a computational tool to segment bacterial and mammalian cells cultured in microfluidic devices and imaged by time-lapse microscopy. The method is based on thresholding and uses the same core functions for both cell types. It allows to segment individual cells in high cell-density microfluidic devices, to quantify fluorescence protein expression over a time-lapse experiment and to track individual cells. ChipSeg enables robust segmentation in external feedback control experiments and can be easily customised for other experimental settings and research aims.

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.

SummaryWe study bothin silicoandin vivothe real-time feedback control of a molecular titration motif that has been earmarked as a fundamental component of antithetic and multicellular feedback control schemes inE. 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 providein silicoevidence 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 anin silicomodel of the motif that can be used as an error computation module in future embedded or multicellular control experiments.

Mouse embryonic stem cells (mESCs) have been shown to exist in three distinct pluripotent states (ground, naïve and primed pluripotent states), depending on culture conditions. External feedback control strategies have been, so far, mainly used to automatically regulate gene expression in bacteria and yeast. Here, we exploit a microfluidics/microscopy platform and segmentation and external feedback control algorithms for the automatic regulation of pluripotency phenotypes in mESCs. We show feasibility of automatically controlling, in living mESCs, levels of an endogenous pluripotency gene, Rex1, through a fluorescent reporter, used as control output, and drugs commonly used to modulate pluripotency (i.e. MEK kinase and Gsk3β inhibitors) as control inputs. Our results will ultimately aid in the derivation of superior protocols for pluripotency maintenance and differentiation of mouse and human stem cells.

While early synthetic biology circuits were implemented in unicellular organisms, new tools for genome editing allow their realization in multicellular systems and bottom-up reconstruction of multicellular phenomena. Still, context dependence and the general lack of robustness challenge the implementation of synthetic gene circuits in mammalian cells; such difficulties can be compensated using control strategies. Here, we propose the first in silico implementation of engineered multicellular control in mammalian cells, through a feedback loop distributed across two cell populations. Our design encompasses a negative feedback loop between the controller and target cell populations, and orthogonal communication devices; also, the controller cell population responds to an external input, considered as the control reference in the system. We adapted an existing Ordinary Differential Equation model for synthetic two-way communication to formalize our design; a computational proof-of-concept of the control strategy feasibility is provided, together with an indication of the possible biological parts to be used for the system experimental implementation. Our approach can be advantageous thanks to its modularity and reduced cell burden; two-way communication-based control could be later used for the implementation of complex multicellular systems with the desired functionality.

Cellular systems have evolved numerous mechanisms to finely control signalling pathway activation and properly respond to changing environmental stimuli. This is underpinned by dynamic spatiotemporal patterns of gene expression. Indeed, in addition to gene transcription and translation regulation, modulation of protein levels, dynamics and localization are also essential checkpoints that govern cell functions. The introduction of tetracycline-inducible promoters has allowed gene expression control using orthogonal small molecules, facilitating rapid and reversible manipulation to study gene function in biological systems. However, differing protein stabilities means this solely transcriptional regulation is insufficient to allow precise ON-OFF dynamics, thus hindering generation of temporal profiles of protein levels seenin vivo. We developed an improved Tet-On based system augmented with conditional destabilising elements at the post-translational level that permits simultaneous control of gene expression and protein stability. Integrating these properties to control expression of a fluorescent protein in mouse Embryonic Stem Cells (mESCs), we found that adding protein stability control allows faster response times to changes in small molecules, fully tunable and enhanced dynamic range, and vastly improved microfluidic-basedin-silicofeedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to finely modulate levels of signalling pathway components in stem cells.

Real-time automatic regulation of gene expression is a key technology for synthetic biology enabling, for example, synthetic circuit components to operate in an optimal range. We show that it is possible to regulate the expression of a reporter protein from the tetracycline-inducible promoter in a population of mammalian cells using principles from automatic control engineering. We demonstrate that the performance of the control experiments improves by moving from a discrete to a continuous actuation. Our automated control platform is an innovative tool to enable dose-response studies of small molecules and investigation of gene dosage effects in disease.

Gene networks and signaling pathways display complex topologies and, as a result, complex nonlinear behaviors. 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 the 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 signaling 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 signaling pathway in single cells; (iii) to build a biohybrid oscillator in single embryonic stem cells by interfacing biological parts with virtualin silicocounterparts. Ultimately, this platform can be used to probe gene networks and signaling 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.