A partially self-regenerating synthetic cell

The creation of a self-replicating synthetic cell is a significant challenge in synthetic biology. While individual components like compartmentalization, metabolism, and DNA replication have been demonstrated, integrating these into a functional whole remains elusive. A self-replicating system, analogous to von Neumann's universal constructor, is a critical requirement. This constructor would use DNA-encoded instructions, external building blocks, and energy to replicate itself. Existing experimental approaches, such as auto-catalytic chemical systems and self-replicating ribozymes, have provided insights, but a fully functional biochemical system remains unrealized. This paper aims to address this challenge by constructing a transcription-translation system capable of sustained self-regeneration of essential protein components. The key challenges include achieving sufficient protein synthesis rates to regenerate components, ensuring functional synthesis of these components, and creating a stable reaction environment. The authors approach these challenges using a continuous transcription-translation reaction within microfluidic reactors, enabling real-time assessment of self-regenerating activity.

The paper references prior work demonstrating progress towards synthetic cell construction, including achievements in compartmentalization, mobility, metabolism, communication, and DNA replication. It highlights von Neumann's concept of a universal constructor as a theoretical framework for self-replication. Previous experimental work with auto-catalytic chemical systems and self-replicating ribozymes is mentioned. The challenges of building a self-regenerating transcription-translation system, namely ensuring sufficient synthesis capacity, functional synthesis, and maintaining a suitable reaction environment, are discussed in relation to existing literature on cell-free protein synthesis systems and their limitations. The authors emphasize the need for optimizing resource allocation and minimizing resource competition within these systems.

The researchers employed a microfluidic device with eight independent reactors, allowing for continuous cell-free reactions and precisely controlled dilution rates. The system uses a modified PURE system (a cell-free protein synthesis system) in which components are added continuously. A "kick-start" method was implemented to initiate regeneration from DNA templates. The process involved three phases: kick-start (using a complete PURE system), self-regeneration (using a PURE system lacking the protein being regenerated), and washout (removal of DNA templates to assess sustained regeneration). Experiments focused on regenerating different aminoacyl-tRNA synthetases (aaRSs), and tRNA polymerase (RNAP). Fluorescent protein (eGFP) expression served as a quantitative readout of protein synthesis capacity. The authors systematically varied DNA concentrations to optimize self-regeneration and map genotype-phenotype relationships. They also developed a computational model to understand resource competition effects. Detailed methods for ribosome purification, PURE system preparation, energy solution preparation, batch in vitro protein expression experiments, SDS-PAGE gel analysis, microfluidic device fabrication, device setup, data acquisition, and data analysis are thoroughly described in the supplementary materials.

The study successfully demonstrated self-regeneration of essential protein components. Successful self-regeneration of two aaRSs (AsRS and LeuRS) was achieved. The DNA input concentration proved critically important for system function; optimal concentrations were identified for robust self-regeneration. High DNA concentrations led to resource competition, reducing eGFP expression. Self-regeneration of T7 RNAP (essential for transcription) was achieved and sustained for over 85 hours at an optimal DNA concentration. The authors successfully achieved simultaneous regeneration of T7 RNAP and multiple aaRSs, demonstrating the feasibility of multi-component regeneration. They found that achieving successful multi-component regeneration requires careful optimization of DNA ratios to minimize resource competition and maximize resource allocation. A computational model confirmed the importance of resource competition in explaining the observed results. The concepts of yield (level of non-essential protein synthesized) and robustness (duration of sustained self-regeneration) were defined to quantify the system’s performance. A Pareto optimal relationship between yield and robustness was observed, indicating a trade-off between maximizing eGFP synthesis and ensuring long-term self-regeneration. Regeneration of up to seven aaRSs simultaneously was achieved, reaching approximately 50% of the theoretically achievable yield while maintaining robust self-regeneration for extended periods.

The findings demonstrate the feasibility of creating a partially self-regenerating synthetic cell, moving closer to the goal of building a fully self-replicating system. The results highlight the importance of resource allocation and competition in these systems, emphasizing the need for precise control of DNA concentrations and ratios for optimal performance. The computational model helps explain the observed experimental behavior, providing a framework for future optimization efforts. The work demonstrates a key step towards the construction of a universal biochemical constructor, mirroring von Neumann’s concept. The study also reveals limitations of current technology; the PURE system’s synthesis rate needs improvement to support full self-replication, and further challenges in ribosome and tRNA synthesis remain.

This research presents a significant advance toward the creation of a self-replicating synthetic cell. The successful partial self-regeneration of essential components, including multiple proteins simultaneously, demonstrates the feasibility of the approach. Future work should focus on improving the synthesis rate of the PURE system, integrating ribosome and tRNA synthesis, and implementing more sophisticated control mechanisms to optimize resource allocation and manage resource competition more effectively. The development of active feedback regulation could enhance system robustness and efficiency. The study emphasizes that a combination of mechanistic understanding and systems-level design principles will be essential for creating a fully self-replicating synthetic cell.

The current system only demonstrates partial self-regeneration; it does not yet include self-replication of all necessary components, such as ribosomes and tRNAs. The synthesis rate of the PURE system needs significant improvement to achieve full self-replication. The current system is also relatively complex, requiring precise control of DNA concentrations and ratios. The computational model is a simplification of the actual system, and additional factors might influence system performance.