Building the genome of a minimal synthetic cell

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.