What is the smallest possible genome that can sustain a viable bacterial cell? Venter and colleagues now report a synthetic genome ('JCVI-syn3.0') that is smaller than any known genome of an autonomously replicating cell and yet can support robust growth when transplanted into Mycoplasma capricolum cells. The 531 kb genome owes its design to a refinement of the Mycoplasma mycoides genome that was guided by transposon mutagenesis screens that identified non-essential genes. The 438 protein-coding genes and 35 RNA-coding genes of JCVI-syn3.0 include those with predicted functions in gene expression (195 genes), the cell membrane (84 genes), metabolism (81 genes), and genome preservation and cell division (36 genes), in addition to 79 genes for which a function could not be predicted. Although not all of the genes were essential for viability, further minimization prevented robust growth, which would preclude the use of cells with genomes smaller than JCVI-syn3.0 as an experimental model.