Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, United States.
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, United States.
Methods Enzymol. 2020;643:203-224. doi: 10.1016/bs.mie.2020.04.023. Epub 2020 May 5.
Knowledge of the distribution of fitness effects (DFE) of mutations is critical to the understanding of protein evolution. Here, we describe methods for large-scale, systematic measurements of the DFE using growth competition and deep mutational scanning. We discuss techniques for producing comprehensive libraries of gene variants as well as provide necessary considerations for designing these experiments. Using these methods, we have constructed libraries containing over 18,000 variants, measured fitness effects of these mutations by deep mutational scanning, and verified the presence of fitness effects in individual variants. Our methods provide a high-throughput protocol for measuring biological fitness effects of mutations and the dependence of fitness effects on the environment.
了解突变的适应度效应（DFE）分布对于理解蛋白质进化至关重要。在这里，我们描述了使用生长竞争和深度突变扫描进行大规模、系统测量 DFE 的方法。我们讨论了产生全面基因变异文库的技术，并为设计这些实验提供了必要的考虑因素。使用这些方法，我们构建了包含超过 18000 个变体的文库，通过深度突变扫描测量这些突变的适应度效应，并验证了单个变体中适应度效应的存在。我们的方法提供了一种高通量的测量突变的生物学适应度效应和适应度效应对环境的依赖性的方案。
