Towards a Synthetic Nucleus: Separating Transcription and Translation in Cell-Free Protein Expression Systems

Citation

Jurado Quiroga, Zoila Estefani (2024) Towards a Synthetic Nucleus: Separating Transcription and Translation in Cell-Free Protein Expression Systems. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/kvq4-6132. https://resolver.caltech.edu/CaltechTHESIS:06032024-141219249

Abstract

Synthetic cells represent the culmination of decades of research aimed at deciphering the intricacies of life at its most basic level. The result of the fusion of biology, chemistry, physics, and engineering, synthetic cells promise to revolutionize biotechnology, medicine, and beyond. This thesis focuses on the ramifications of incorporating a synthetic nucleus within a synthetic cell.

To experimentally study transcription and translation, we use a commercially available cell-free protein expression system comprising all the purified proteins essential for protein production (PURE), along with a fluorescent RNA aptamer--malachite green aptamer (MGapt), and a green fluorescent protein (deGFP). We observed that the chemical composition of the PURE system significantly impacts MGapt fluorescence, leading to inaccurate RNA calculations. We identify the reducing agent, dithiothreitol (DTT), to address this challenge as a crucial chemical affecting MGapt fluorescence. We propose a model that can reliably model MGapt measurements in commercial PURE. This investigation illuminates the intricate dynamics of MGapt in PURE and emphasizes the necessity of accounting for environmental factors in RNA measurements employing aptamers.

Subsequently, to advance our understanding of a synthetic nucleus and analyze the effects of separating transcription and translation in a cell-free protein expression, we propose and validate a chemical reaction network model for transcription (TX) in PURE. Additionally, we used open-source software to expand an existing translation (TL) model for any arbitrary DNA sequence to create a nearly complete model of TX-TL in PURE. Leveraging this model, we investigate the effect of introducing a synthetic nucleus by modulating the RNA diffusion rate and resource allocation. This detailed model showcases our capability to comprehensively model protein expression in PURE, enabling insights into the efficacy of segregating transcription and translation processes within the artificial cell environment. Lastly, we provide a perspective on the future of synthetic cells with an artificial nucleus and propose further steps to develop the proposed synthetic nucleus model.

Thesis Files