Autocatalytic selection of gene functions in vitro

The integration of biological functions into a single operating system is considered a major challenge in the construction of a synthetic cell1-3. We present autocatalytic selection (ACS) of gene functions as a driver for integrating biological modules in vitro. A gene of interest (GOI) is introduced into a minimal DNA self-replicator based on the {phi}29 replication machinery4 and the function of the GOI is linked to transcription, translation or DNA replication through a positive feedback loop. As the encoded function eventually promotes DNA self-replication, the gene variants with greater activity are selected. Using different coupling mechanisms, we demonstrate ACS of three functions: transcription, synthesis of a deoxynucleoside triphosphate for DNA replication, and {beta}-galactosidase activity. The latter example illustrates how a function that is unrelated to the Central Dogma can be selected. This work paves the way for ACS-driven Darwinian evolution of virtually any biomolecule in vitro, streamlining the construction of increasingly complex synthetic cells as well as the engineering of biotechnologically relevant enzymes.