Rapid advances in DNA synthesis techniques have made it possible to engineer diverse genomic elements, pathways, and whole genomes, providing new insights into design and analysis of systems. The synthetic yeast genome project, Sc2.0 is well on its way with six of the first synthetic Saccharomyces cerevisiae chromosomes completed. Undergraduate students provide a workforce for synthesis and assembly for some of these chromosomes, though a wide variety of assembly schemes are employed by the various groups building chromosomes. The synthetic genome features several systemic modifications, including TAG/TAA stop-codon swaps, deletion of subtelomeric regions, introns, tRNA genes, transposons and silent mating loci. As well, strategically placed loxPsym sites enable genome restructuring using an inducible evolution system termed SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution).
SCRaMbLE can be used as a novel method of mutagenesis, capable of generating millions of variants leading to complex genotypes and a variety of phenotypes. The fully synthetic yeast genome opens the door to a new type of combinatorial genetics based on variations in gene content and copy number, rather than base changes. We also describe supernumerary designer “neochromosomes” that add new functionalities to cells such as humanized pathways and complexes, and general approaches to engineering of karyotype. Finally, we have automated many steps in our big DNA synthesis pipeline, opening the door to massively parallel big DNA assembly.