Building a synthetic cell from scratch is a long-term goal for bottom-up synthetic biology. Here, I have presented my work on the implementation of the self-regeneration feature for a synthetic cell building exercise. Self-regeneration refers to the ability of the system to renew all the molecular contents present at the required concentration. The PURE (Protein synthesis Using Recombinant Elements) system, a reconstituted cell-free transcription and translation system, is used as a model system for exploring the self-regeneration feature. The PURE system consists of 36 non-ribosomal proteins involved in transcription (T7RNAP), translation (10 proteins), aminoacylation (20 proteins), and energy regeneration (5 proteins) modules for protein expression.

Chapter 2 of the thesis reviews the origins of the cell-free systems, our current understanding of the genetic code, explains the role of AARS and tRNA in molecular biology, and details the biochemistry underpinning the interaction between AARSs, tRNA, and their substrates. A summary of the synthesis and engineering of tRNA and AARS molecules in the cell-free conditions is presented. An overview of various aminoacylation strategies used in cell-free conditions is provided.

Chapter 3 details the PURE optimization research on non-ribosomal PURE proteins. A key limitation for complete self-regeneration of all 36 non-ribosomal PURE proteins is the low synthesis rate and high protein concentration in the system. The concentration of each non-ribosomal PURE protein was optimized, resulting in the PURE v3 system with a 78% reduction in total non-ribosomal protein concentration from 920.6 µg/ml to 201.56 µg/ml, while doubling the protein synthesis efficiency. The diluted PURE formulations from PURE v3 gained much with the addition of a crowding agent, such as dextran-70, which doubled the synthesis rate and final yield of the protein expression. This work laid the possibility of complete self-regeneration of all non-ribosomal PURE proteins.

Chapter 4 details the efforts to implement self-regeneration of non-ribosomal PURE proteins using the serial transfer experimental setup. The transfer volume, transfer interval, number of transfers, and template concentration of non-ribosomal PURE proteins and eGFP reporter were optimized. The self-regeneration of proteins was tracked using either native eGFP fluorescence or acquired fluorescence from Bodipy tRNA lysine. The results from self-regeneration experiments were inconsistent with previous observations and met with difficulties in implementing this feature. The possible impact of the PURE reaction background, cross-contamination of PURE proteins, and choice of experimental conditions on self-regeneration is addressed.

Chapter 5 demonstrates the utility of the PURE system as a screening platform for drug compounds. In the fight against rising antimicrobial resistance (AMR), novel tools and strategies such as Atomnet®, a deep convolutional neural network, and biochemical assays, targeted towards specific drug: target interactions, were used to screen novel drug components against the FusA1 protein, involved in the protein translation process. A modified PURE system was developed to study the effect of fusidic acid, enamine compounds predicted using Atomnet, and MGC compounds synthesized as fusidic acid analogs on FusA1 WT and mutant H462Y protein.