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Smart Lab-On-Chips for the Real -Time Control of Cells

Periodic Reporting for period 2 - SmartCells (Smart Lab-On-Chips for the Real -Time Control of Cells)

Reporting period: 2018-10-01 to 2020-03-31

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. For example, In the long-term, researchers will create synthetic cells that could, for example, be introduced into the human gut microbiome to improve human health; such approaches have the potential to radically improve everyday life. 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. Here, we will combine microfluidics, optogenetics, structured illumination, inference methods and control algorithm into such a real time control device of gene expression for yeast cells. We will then study 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 aim at: (1) Understanding the potential and limits of such a control method. We will ask to what extent robust control can be achieved at the single cell level over a broad range of operating conditions; (2) Taking control of complex circuits. In particular, we will take control of key genes of the large regulatory network in charge of yeast adaptation to osmotic stress and dissect their roles in setting the mechano-biology properties of yeast; (3) Taking control of multicellular systems. We will control the collective dynamics of a population of cells via single cell control at selected locations. Taken together, these actions will establish 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. 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 so far, 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. This will allow us to try to control key genes of this response and use it to drive the cell physiology while it is responding to adverse stresses. Finally, we have also set up a novel microfluidic device and light responsive strains that will be needed to explore the applications of multiple, parallel single cell control to drive a consortium of cells with optogenetic."
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 gene. At the end of the SmartCell project, we expect to 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. Second, we will demonstrate its applicability over increasingly complex biological systems with a focus on the establishment of a theoretical framework of control theory for biology. Said differently, we will explore the potentials and limitations of real time control that are intrinsically linked to the biological machinery and that must be taken into account to conceive synthetic biology applications that can be put into action outside of the laboratories.
Principle of real time control of genetic circuits