Advancing Cell-Free Synthetic Biology: From sustained PURE protein synthesis to an isothermal one-pot DNA replication system

One of the goals of synthetic biology is the development of an artificial cell. Building an artificial cell from scratch will provide a deeper understanding of fundamental mechanisms and models in biology and promises to contribute towards building novel platforms that can be leveraged to drive bioengineering innovation. There is ongoing debate on what a synthetic cell looks like in detail; here we define an artificial cell as a system that is able to self-replicate and evolve. Self-replication entails the capability of renewing all of the components of the cellular machinery and replicating its genetic blueprint. If errors in the copying of the genetic blueprint can occur and are propagated, the system is in principle able to undergo Darwinian evolution. One scheme for realizing such an artificial cell is based on transcription-translation (TX-TL). In turn, the most widely-used practical implementation of the TX-TL machinery is the well-defined PURE cell-free system, in which case the genetic blueprint is stored in the form of DNA. Cell-free systems do not only offer a possible foundation for a future synthetic cell, but also provide a powerful platform for innovation in the area of bioengineering. This thesis first reviews and compares existing cell-free platforms, while highlighting opportunities for applications to address a multitude of scientific questions. Chapter 3 introduces a protocol for setting up a OnePot PURE system that is cost-effective and can be prepared within one week. The OnePot PURE system thereby addresses a key shortcoming of the conventional PURE system: its high cost currently impedes its widespread adoption. Our approach streamlines the process of protein purification of all 36 non-ribosomal proteins to one purification step, thereby saving time and costs. Chapter 4 details the development of a microfluidic chemostat, augmented with semi-permeable membranes, which addresses a second limitation of the PURE system: its capacity for protein synthesis is at least 25x too low to regenerate all of its components. Implementing dialysis on our chemostat resulted in significantly extended protein synthesis, leading to a 6-fold increase in protein levels at steady-state. Chapter 5 describes C2CAplus, a one-pot isothermal Circle-to-Circle DNA amplification (C2CA) system. Rolling circle amplification (RCA) is a DNA amplification method that has found widespread usage in biosensing applications and is considered the most promising approach towards developing a DNA replication system for a synthetic cell. However, standard RCA generates linear, multimeric product from circular input DNA, which is not ideal for the construction of an artificial cell. C2CAplus implements a recircularization method employing restriction digest and ligation. The method is highly sensitive and efficient, and the amplified DNA product can be seen with the naked eye, suggesting that C2CAplus also holds promise for use as a biosensor. Across these chapters,