Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-continuous evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a continuous flow apparatus and the performance and analysis of evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.

定向进化将达尔文进化论的原理应用于实验室环境，是生成具有多样化和定制特性的生物分子的强大策略。该技术可以使用连续进化以高效的方式实现，这使得定向进化的步骤能够在研究人员干预最少的情况下无缝地在许多连续代中进行。噬菌体辅助连续进化（PACE）通过将达尔文进化的步骤映射到噬菌体生命周期来实现细菌的连续定向进化，并允许定向进化以比传统方法更快的时间尺度发生。该协议提供了有关使用 PACE 和噬菌体辅助非连续进化 (PANCE) 进化蛋白质的详细说明，并包括有关选择噬菌体和宿主细胞的制备、连续流装置的组装以及进化实验的性能和分析的信息。该协议可以在短短 2 周内完成，在单个 PACE 实验中完成 100 多轮进化（突变、选择和复制的完整循环）。