A cell-free bacteriophage synthesis system for directed evolution.

Efficient phage production has always been an urgent need in fields such as drug discovery, disease treatment, and gene evolution. To meet this demand, we constructed a robust cell-free synthesis system for generating M13 phage by simplifying its genome, enabling a three-times faster efficiency compared with the traditional method in vivo. We further developed a cell-free directed evolution system in droplets, comprising a modified helper plasmid (ΔPS-ΔgIII-ΔgVI) and the simplified M13 genome-carrying gene mutation library. This system was greatly improved when coupled with fluorescence-activated droplet sorting (FADS). We successfully evolved the T7 RNA polymerase (RNAP), achieving a twofold higher activity to read through the T7 terminator. Moreover, we evolved the tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity to read through the stop codon UAG.