Periodic Reporting for period 5 - SmartCells (Smart Lab-On-Chips for the Real -Time Control of Cells)
Période du rapport: 2022-08-01 au 2023-09-30
Cells are complex, autonomous genetic machines with rich information processing capabilities. Synthetic Biology builds on these properties to design novel, synthetic genetic programs in cells with the aim of benefiting humans in their everyday life. For example, researchers are imagining bacterial therapeutic cells that could, for example, be used to improve human health; More generally, synthetic biology and the “made by biology” approaches have the potential to radically improve everyday life. Yet, this will require the ability to safely and robustly controlling such synthetic cells. This is a tremendous challenge for synthetic biologists, as the robustness of any circuit is limited by their high dependence on the cellular host machinery and the fundamental stochastic nature of gene expression. Taking inspiration from physics and engineering we have imagined a computer-based feedback loop that can remotely, in real-time, control the state of a synthetic genetic program running in cells. In this project, we combined microfluidics, optogenetics, inference methods and control algorithms into a real time control device of gene expression for cells. We notably studied how cells can be controlled at different scales and with increasing levels of complexity from a simple circuit to a simple multicellular ecosystem. Specifically, we showed that simple protein expression can be controlled in yeast cells, (2) we demonstrated that complex circuits, such as bistable synthetic genetic circuit, can also be controlled robustly and designed novel strategies to control them. We also took control of key genes that are used by cells to perform complex adapative response, such as adaptation to osmotic stress in yeast. Finally, we explored the possibility to control multicellular assemblies through the control of its individual components. We demonstrated that optogenetics can be used to trigger the behaviour of cells at selected location and studied its impact on the overall population. Taken together, these actions established a solid scientific and technological foundations of a novel research area, called Cybergenetics, combining physics, engineering and synthetic biology to take control of living systems. More globally, we envision that this project will provide the first steps towards the design and study of hybrid bio-robotic systems in which machine and living systems feedback on each other to provide robust behaviors that could be applied to bioengineering and health technological and scientific challenges.
Since the beginning of the project, we have made significant progress on two main aspects. First, we have now built a technological framework that allows us to control cells in real time, based on fluorescence microscopy images that are acquired periodically by a microscope. This required to work on synthetic biology, on improving microfluidic devices and image analysis methods. As of now, every actions, decisions and computations are done in real time, effectively creating a closed feedback loop that pilot, robustly the behavior (e.g. fluorescence state) of cells for extended period of time. This framework is still being developed to improve its efficiency and its versatility. It has also open the possibility to develop a smart microscopy open source software that can be used to design advanced, conditional based, time lapse microscopy experiments, a critical need for cell biologist. We also developed optogenetics tools to control gene expression and other processes through light at the single cell level.
In addition, we have made scientific advances to apply real time control of gene expression for increasingly complex biological systems. An important outcome of the project is the demonstration that it is possible to control unstable gene regulatory network with our method. We showed that a genetic "toggle switch" that is intrinsically bistable can be dynamically controlled, and maintained near its unstable equilibrium by real time modulation of the concentrations of two small molecules in the cell environment. This is analog to the control of the inverted pendulum into its unstable upward position, a hallmark of control theory for electro-mechanical system. Going towards increasing biological complexity, we have also made important progress on our understanding of the genetic network that drives the response to osmotic stress in yeast. Indeed, we performed a complete, quantitative study of how the osmotic stress regulatory pathway can process information (a prerequisite of setting up control strategies) in function of the metabolic environment. This quantitative study took us on an expected line of research in which we showed that the presence or absence of glucose can drastically change the signaling and transcriptional response of yeast to osmotic stress, putting back metabolic activity as a main regulator of stress response into the picture of Cybergenetics. Finally, we explored with different examples that involve cell-cell communication the possibility to control an assembly of cells using only a limited number of observable/ actionable genes and relying on optogenetic activation to control selected cells within a larger assembly.
In addition, we have made scientific advances to apply real time control of gene expression for increasingly complex biological systems. An important outcome of the project is the demonstration that it is possible to control unstable gene regulatory network with our method. We showed that a genetic "toggle switch" that is intrinsically bistable can be dynamically controlled, and maintained near its unstable equilibrium by real time modulation of the concentrations of two small molecules in the cell environment. This is analog to the control of the inverted pendulum into its unstable upward position, a hallmark of control theory for electro-mechanical system. Going towards increasing biological complexity, we have also made important progress on our understanding of the genetic network that drives the response to osmotic stress in yeast. Indeed, we performed a complete, quantitative study of how the osmotic stress regulatory pathway can process information (a prerequisite of setting up control strategies) in function of the metabolic environment. This quantitative study took us on an expected line of research in which we showed that the presence or absence of glucose can drastically change the signaling and transcriptional response of yeast to osmotic stress, putting back metabolic activity as a main regulator of stress response into the picture of Cybergenetics. Finally, we explored with different examples that involve cell-cell communication the possibility to control an assembly of cells using only a limited number of observable/ actionable genes and relying on optogenetic activation to control selected cells within a larger assembly.
Achieving the real time control of cellular functions will open the possibility to precisely probe important biological phenomenon based on the dynamic, controlled perturbation of well-chosen genes. At the end of the SmartCell project, we have made significant, beyond the state of the art, advances on two fronts. First regarding the development of a user friendly, versatile technological framework to perform real time control for various cellular systems and in various biological context. This is now an ERC POC project dedicated to perform smart microscopy experiments. Second, we demonstrated real time control applicability over increasingly complex biological systems, on concrete biological examples that can serve as foundational example for the future development of what is now called Cybergenetics.