Cell-free protein synthesis system as a prototyping platform for mammalian synthetic biology

Abstract

As the field of Synthetic Biology is expanding, there is an increasing need for methods to develop new modes of synthetic gene regulation. A demand for measurement methodologies which provide results that can be interpreted outside a specific context and used to design larger composite devices in diverse cell types is especially urgent. The motivation of my PhD research was to establish new prototyping tools to measure gene regulation, specifically for mammalian applications. Two different experimental approaches were explored. At first, a tissue culture based method was adapted to measure gene expression regulation. However, it was found that this method would not scale to testing many variants, and alternatives needed to be sought. The results from characterising an RNA binding protein which can repress translation (L7Ae) in cells were used as a reference point to compare later obtained results using the second cell-free measurement approach. The hypothesis of this thesis comprises of establishing a new cell-free protein synthesis (CFPS) method as a prototyping tool for mammalian gene expression. The sections involve an initial proof-of-concept phase, the development of a semi-automated workflow, discovery of novel findings using the method, and preliminary work with the aim of establishing a mathematical modelling framework. The hypothesis that CFPS is an appropriate model for gene expression in mammalian cells is proven across the biochemical analysis of transcription and translation steps. The potential of CFPS was demonstrated through prototyping diverse modes of gene regulation, including transcription and translation regulation. Specifically, T7 constitutive promoter variants, IRES constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression were characterised. Liquid handling technology was used to automate reaction assembly and software tools were developed to aid the experimental process throughout design, implementation and data analysis. A graphical user interface was designed to enable wide use of the method. The robustness of the method was established through comparison of repeated experiments. In the last section, the feasibility to measure RNA production in real time in CFPS was established. A mathematical model of bacterial CFPS was adapted to the mammalian context, and preliminary parameter inference was carried out, based on experimental data. Overall, the results contribute to the understanding of the biochemistry of L7Ae, CRISPR/Cas9 and transcription in mammalian systems. The established automated methods will impact mammalian synthetic biology by enabling further work at a faster scale than would be possible using traditional experimental approaches.