Geometric Control of Bio-Machine Assembly By a Genetic Program on a Chip

From viruses to ribosomes, large macromolecular machines self-assemble inside cells in a highly orchestrated and efficient manner, regulated by the spatial organization of genes in operons and clusters, by gene expression foci and by the crowded cytoplasm. We seek to define a cell-free architecture of DNA programs to achieve geometric self-organization of protein assembly pathways. We present an emerging surface approach to recreate and investigate bio-assembly processes crucial to the creation of a self-replicating artificial cell. RNA and protein machine parts are synthesized in a cell-free reaction from high-density surface-immobilized gene compartments and self-assemble into large complexes on nearby surface traps. The DNA compartments localize the gene-expression machinery, creating a local source of RNAs and proteins, coupling synthesis to assembly. Surface trapping of assembly products provides structural scaffolding and a highly sensitive fluorescent imaging in time and space. Using this methodology, we recreate the assembly pathway of both T4 bacteriophage baseplates and E. coli 30S ribosomal subunits starting from only their genes, realizing a cell-free scenario for ribosomes making ribosomes.