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Directed Evolution Library Creation: Methods and Protocols, Second Edition presents user-friendly protocols for both proven strategies and cutting-edge approaches for the creation of mutant gene libraries for directed evolution. As well as experimental methods, information on current computational approaches is provided in a user-friendly format that will allow researchers to make informed choices without needing to comprehend the full technical details of each algorithm. Directed evolution has become a fundamental approach for engineering proteins to enhance activity and explore structure-function relationships, and has supported the rapid development of the field of synthetic biology over the last decade. Divided into three convenient sections, topics include point mutagenesis strategies, recombinatorial methods wherein genetic diversity is sourced from multiple parental genes that are combined via either homology-dependent or -independent techniques and a variety of computational methods to guide the design and analysis of mutant libraries. Written in the 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 protocols and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible, Directed Evolution Library Creation: Methods and Protocols, Second Edition will serve as a reliable manual for both novice and experienced protein engineers and synthetic biologists and will enable further technical innovation and the exploitation of directed evolution for a deeper understanding of protein design and function.
Table des matières
Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution.- Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesis.- Random Mutagenesis by Error-Prone Pol Plasmid Replication in Escherichia coli.- The Sequence Saturation Mutagenesis (SeSaM) Method.- Generation of Effective Libraries by Neutral Drift.- Site Saturation Mutagenesis.- Iterative Saturation Mutagenesis (ISM): A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution.- Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides During Gene Shuffling (ISOR).- OmniChange: Simultaneous Site-Saturation of up to Five Codons.- Random Insertional-Deletional Strand Exchange Mutagenesis (RAISE): A Simple Method for Generating Random Insertion and Deletion Mutations.- Transposon-Based Approaches for Generating Novel Molecular Diversity during Directed Evolution.- Restriction Enzyme-Mediated DNA Family Shuffling.- Assembly of Designed Oligonucleotides (ADO): A Useful Tool in Synthetic Biology for Creating High Quality Combinatorial DNA Libraries.- One-Pot, Simple Methodology for Cassette Randomization and Recombination for Focused Directed Evolution (OSCARR).- USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homology.- ITCHY: Incremental Truncation for the Creation of Hybrid Enzymes.- Generating Random Circular Permutation Libraries.- Probabilistic Methods in Directed Evolution: Library Size, Mutation Rate and Diversity.- The Mutagenesis Assistant Program.- Computational Tools for Designing Smart Libraries.- Computational Tools for Directed Evolution: A Comparison of Prospective and Retrospective Strategies.- Designing Libraries of Chimeric Proteins Using SCHEMA Recombination and RASPP.- Non-Contiguous SCHEMA Protein Recombination.- Engineering Proteins by Reconstructing Evolutionary Adaptive Paths.
Résumé
Directed Evolution Library Creation: Methods and Protocols, Second Edition presents user-friendly protocols for both proven strategies and cutting-edge approaches for the creation of mutant gene libraries for directed evolution. As well as experimental methods, information on current computational approaches is provided in a user-friendly format that will allow researchers to make informed choices without needing to comprehend the full technical details of each algorithm. Directed evolution has become a fundamental approach for engineering proteins to enhance activity and explore structure-function relationships, and has supported the rapid development of the field of synthetic biology over the last decade. Divided into three convenient sections, topics include point mutagenesis strategies, recombinatorial methods wherein genetic diversity is sourced from multiple parental genes that are combined via either homology-dependent or -independent techniques and a variety of computational methods to guide the design and analysis of mutant libraries. Written in the 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 protocols and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible, Directed Evolution Library Creation: Methods and Protocols, Second Edition will serve as a reliable manual for both novice and experienced protein engineers and synthetic biologists and will enable further technical innovation and the exploitation of directed evolution for a deeper understanding of protein design and function.
