In Vitro� Ribosome Synthesis and Evolution through Ribosome Display

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent ribosomes (iSAT) enables the rapid generation of large libraries of ribosome variants in a cell-free environment. Here we combine the iSAT system with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of ribosome variants. We further demonstrate the prevalence of positive epistasis in successful genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.