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.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
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.