Autonomous biogenesis of all thirty proteins of the Escherichia coli translation machinery.

The cell-free biogenesis of the protein translation machinery is essential for the creation of a self-regenerating synthetic cell. Here, we demonstrate the autonomous and simultaneous biogenesis of all thirty proteins of the translation machinery of E. coli in a reconstituted transcription-translation system. We first establish self-regeneration of every translation protein by determining the threshold concentration required for its own synthesis from a synthetic gene coding for the protein, thereby demonstrating the functionality of all nascent proteins, separately. Simultaneous biogenesis of multiple translation proteins at their threshold results in delayed synthesis below detection levels. To achieve self-regeneration of multiple translation proteins, we induce boundary-free compartmentalization of the reaction by immobilizing the genes on a surface at high density. The co-localization of genes, molecular machinery, messenger RNA and nascent proteins at the surface create sufficient conditions to catalyze the simultaneous self-regeneration of sub-groups up to all thirty translation proteins, as measured by total internal reflection fluorescence on the surface. Our approach provides mechanistic insight and presents a general methodology for the biogenesis of cellular machines toward autonomous synthetic systems.