Sakurai, A.; Shoji, K.; Ichihashi, N.

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.