Towards an artificial cell: Development of a system which self-regenerates the protein components of the PURE system in microfluidic reactors

Thanks to recent advancements in synthetic biology, the dream of creating a synthetic cell has become feasible. However, due to its inherent complexity, one of the fundamental functions of all living systems, i.e., self-replication, remains to be introduced. The development of a system capable of self-regeneration faces several challenges, as the system needs to be able to functionally synthesize all of its components at sufficient capacity in an environment that allows continuous and sustained regeneration. In this work, we have developed a system coupling a microfluidic platform with cell-free systems, which provides a viable approach for developing and optimizing self-regeneration at non-equilibrium conditions. Reconstituted transcription-translation systems are a viable starting point for achieving a self-regeneration system. Therefore, in this work, we begin by presenting a simple, robust, and low-cost production method for the cell-free system called protein synthesis using recombinant elements (PURE). Our approach relies on streamlining protein purification by coculturing and co-purification. We show that our "OnePot" method allows for minimizing time and labor requirements while preserving the versatility and purity of the system. Moreover, we demonstrate that the OnePot PURE system can achieve a similar protein synthesis yield to a commercial PURE system, which leads to a 14-fold improvement in cost-normalized protein synthesis yield over existing PURE systems with similar composition.Living organisms continuously exchange energy and matter with their environment. Similarly, one of the requirements of continuous and sustained regeneration is a life-like non-equilibrium environment. Therefore, we developed an improved microfluidic chemostat with fluidically hard-coded dilution fractions defined by the reactor geometry, which enable long-term steady-state reactions. We employed the introduced microfluidic platform in combination with the PURE systems to study self-regeneration. We demonstrated that the system can regenerate proteins essential for transcription and translation from DNA templates and that simultaneous self-regeneration of multiple proteins is sustainable in the system. Moreover, in combination with computational modeling, we showed that minimizing resource competition and optimizing resource allocation is critically important for achieving robust system functions. Lastly, we developed a microfluidic platform with an integrated hydrogel membrane with adjustable permeability. The integrated membranes separate transcription-translation machinery from the feeding solution of small molecular components, which can diffuse into the reactor through the membranes without diluting the machinery. Utilizing the dialysis-based continuous-exchange reaction, we extended the protein synthesis beyond traditional batch conditions.