Continuous directed evolution rapidly implements cycles of mutagenesis, selection, and replication to accelerate protein engineering. However, individual experiments are typically cumbersome, reagent-intensive, and require manual readjustment, limiting the number of evolutionary trajectories that can be explored. We report the design and validation of Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE), an automation platform for the continuous directed evolution of biomolecules that enables real-time activitydependent reporter and absorbance monitoring of up to 96 parallel evolution experiments. We use this platform to characterize the evolutionary stochasticity of T7 RNA polymerase evolution, conserve precious reagents with miniaturized evolution volumes during evolution of aminoacyl-tRNA synthetases, and perform a massively parallel evolution of diverse candidate quadruplet tRNAs. Finally, we implement a feedback control system that autonomously modifies the selection strength in response to real-time fitness measurements. By addressing many of the limitations of previous methods within a single platform, PRANCE simultaneously enables multiplexed, miniaturized, and feedback-controlled continuous directed evolution.
Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency
