Programming a synthetic out-of- equilibrium reaction network within proteinosomes

Abstract

Life's persistence is underpinned by dynamic biological systems operating across a variety of scales and complexities. Central to these systems is the ability of cells to dynamically adapt to external stimuli, fostering complex and coordinated behavior within cellular communities. Such complex behaviors emerge from networks of biochemical reactions that govern information processing both within individual cells and across cell populations. This is a universal trait, manifesting from bacterial quorum sensing to the signalling pathways integral to embryonic development. Unravelling the intricate structure of network-driven population behaviors necessitates an understanding of how the interaction of compartmentalized information processing and intercellular communication influences dynamic systems. In studying dynamic biological systems, methodologies vary widely, encompassing animal models, organoids, cell cultures, and extending to synthetic biology, each representing a step toward less biological complexity and providing unique insights while presenting their own sets of challenges. Synthetic biology, in particular, offers a focused approach to dissecting these complex systems. Initial research in this field has replicated complex cellular behaviours with compartmentalized, non-enzymatic reactions. Yet, there remains an essential goal to develop enzyme-driven dynamic systems that more accurately mirror the cyclical processes of degradation and synthesis seen in living organisms. This study aims to fill this gap by reconstructing a minimal dynamic system to investigate emergent behaviours through enzyme-based reactions. Specifically, it focuses on the compartmentalized processing of chemical information, activated by diffusible small molecules, and examines its influence on complex intercellular network formation. Employing synthetic biology, the study utilizes microfluidic-assembled droplets—termed proteinosomes—to create populations of engineered synthetic cells. DNA reactions are encapsulated within these proteinosomes populations to enable specific biochemical reactions via controlled diffusion of molecules, allowing the creation of interconnected networks of reactions. In this work, these reactions within proteinosomes are primers activated and enzyme-driven isothermal DNA reaction, known as PEN-DNA reactions. This process involves a short oligonucleotide primer initiating replication from a longer DNA template, producing two shorter oligonucleotides that can diffuse in solution or, as in this case, through the proteinosome’s membrane. The compartmentalization of the reactions leads to kinetic behaviors that are not present in buffer solutions. For instance, autocatalytic reactions within the proteinosomes occurred at rates comparable to those in buffer solutions but with significantly less DNA template. Reaction rates were found to be markedly higher in relation to template concentration than those in buffer solution. This can be linked to the proteinosomes' selective containment of template DNA while allowing primer DNA to diffuse away from the reaction site. Thus, the system is well-suited for creating reaction networks that leverage compartmentalized information processing with communication between compartments. The effectiveness of this platform was demonstrated by first establishing a linear communication between two proteinosome populations, each containing distinct DNA reactions. The network's complexity was then enhanced by incorporating a negative feedback loop, resulting in oscillatory behavior between DNA reactions within the proteinosome populations. This work not only presents a reliable method for constructing compartmentalized reaction networks but also allows for the examination of how spatial localization and passive diffusion affect reaction rates. These synthetic cells facilitate the examination of reaction dynamics within compartments, including the establishment of an oscillatory communication network displaying emergent behaviours. The findings suggest that bottom-up methods can be employed to develop synthetic systems that mimic reaction-diffusion across networks, driven by selective compartmentalization and cellular communication.:Contents 1. ACKNOWLEDGEMENTS ............................................................................................ 3 2. ABSTRACT .................................................................................................................. 5 3. ZUSAMMENFASSUNG ............................................................................................... 7 4. RELATED PUBLICATIONS ....................................................................................... 14 5. ABBREVIATION LIST ................................................................................................ 16 7. CHAPTER 1: INTRODUCTION ................................................................................. 8. CHAPTER 2: PREPARATION OF PEN-DNA REACTION IN PROTEINOSOMES .. 47 9. CHAPTER 3: LINEAR COMMUNICATION BETWEEN DNA TEMPLATES VIA PEN- DNA REACTION IN PROTEINOSOMES ....................................................................... 80 10. CHAPTER 4: PREDATOR-PREY PEN-DNA REACTION WITHIN PROTEINOSOMES POPULATION ............................................................................... 90 11. CHAPTER 5: DISCUSSION ................................................................................. 108 12. CHAPTER 6: ........................................................................................................ 116 13. BIBLIOGRAFY ..................................................................................................... 13