Mechanical and Civil Engineering Seminar: PhD Thesis Defense

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. In this thesis, we focus 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. To address this challenge, we identify the reducing agent, dithiothreitol (DTT), as a crucial chemical affecting MGapt fluorescent and propose a model that can reliably model MGapt measurements. 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 advanced 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.

Please join us in person in 147 Noyes or via Zoom https://caltech.zoom.us/j/87438991225