The research in this dissertation was conducted within the framework of the Building a Synthetic Cell (BaSyC) consortium, which aims to construct a minimal synthetic cell. This is a simple cell containing a minimal set of genes required and sufficient to exhibit the fundamental properties of life. Achieving this goal will deepen our understanding of the essential principles underlying cellular life. This dissertation explores a crucial step towards the realization of a minimal synthetic cell: the de novo design and assembly of its genome. Minimal genome assembly is performed by transformation of the yeast Saccharomyces cerevisiae with DNA fragments, which are assembled into a chromosome by the yeast’s native homologous recombination machinery. The assembled chromosomes are then isolated from yeast and tested for expression in vitro. With the methodologies developed in this dissertation for the design, assembly, screening, isolation, and characterization of synthetic chromosomes for the minimal cell, as well as the constructed chromosome prototypes, we have paved the way for future engineering of minimal genomes and integration of functional modules in synthetic cells.

Life, the most complex and admirable machine that one could think of has evolved over billions of years to display a beautiful variety of mechanisms that keep cells adapting, self-maintaining, reproducing, and evolving. If we think about it, what is this magic? What are the mechanisms behind life’s origins and wonderful coordination? Attracted by these intricates, different scientific disciplines have for long studied all life’s scales to grasp the fundamental principles of life. In particular, the synthetic biology field has set the goal of discerning life until the point that a minimal synthetic cell can be fully recreated in a controlled laboratory set-up. Synthetic cells, modular enough to be crafted by scientists, could not only reveal fundamental insights of how life works, but can also help unlock great biotechnological applications that lie beyond the reach of our current technologies and understanding of life. In this thesis, we delve into how in vitro evolution, module integration, and high throughput characterization are valuable steps to consider for accelerating the bottom-up assembly of artificial cells.