Designing Overlap Extension PCR Primers for Protein Mutagenesis: A Programmatic Approach -- Recombination of Single Beneficial Substitutions Obtained from Protein Engineering by Computer-Assisted Recombination (CompassR) -- Non-Degenerate Saturation Mutagenesis: Library Construction and Analysis via MAX and ProxiMAX Randomization -- Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries -- SpeedyGenesXL An Automated, High-Throughput Platform for the Preparation of Bespoke Ultra-Large Variant Libraries for Directed Evolution -- Facile Assembly of Combinatorial Mutagenesis Libraries using Nicking Mutagenesis -- GeneORator: An Efficient Method for the Systematic Mutagenesis of Entire Genes -- Rapid Cloning of Random Mutagenesis Libraries using PTO-QuickStep -- Construction of Strong Promoters by Assembling Sigma Factor Binding Motifs -- Application of Restriction Free (RF) Cloning in Circular Permutation -- Site-Directed Mutagenesis Method Mediated by Cas9 -- Directed Evolution of Transcription Factor-Based Biosensors for Altered Effector Specificity -- A Screening Method for P450 BM3 Mutant Libraries using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds -- Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method -- Learning Strategies in Protein Directed Evolution.

This volume explores the latest techniques used by researchers to study directed evolution (DE) at each stage of the Design-Build-Test-Learn cycle. Chapters in this book cover topics such as designing overlap extension PCR primers for protein mutagenesis; antha-guided automation of Darwin assembly for the construction of bespoke gene libraries; rapid cloning of random mutagenesis libraries using PTO-Quickstep; and DE of glycosyltransferases by a single-cell screening method. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Directed Evolution: Methods and Protocols is a valuable resource for scientists and researchers who are interested in learning more about this field and incorporating these studies into new experimental workflows.