Directed Evolution: Methods and Protocols

ISBN-13: 9781071621516 / Angielski / Twarda / 2022

Currin, Andrew

Opis

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.

Spis treści

Preface…
Table of Contents…
Contributing Authors…

1. Designing Overlap Extension PCR Primers for Protein Mutagenesis: A Programmatic Approach
Xiaofang Huang, Liangting Xu, Chuyun Bi, Zhao Lili, Zhang Limei, Xuanyang Chen, Shiqian Qi, and Shiqiang Lin

2. Recombination of Single Beneficial Substitutions Obtained from Protein Engineering by Computer-Assisted Recombination (CompassR)
Haiyang Cui, Mehdi D. Davari, and Ulrich Schwaneberg

3. Non-Degenerate Saturation Mutagenesis: Library Construction and Analysis via MAX and ProxiMAX Randomization
Anupama Chembath, Ben P.G. Wagstaffe, Mohammed Ashraf, Marta M. Ferreira Amaral, Laura Frigotto, and Anna V. Hine

4. Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries
P. Handal-Marquez, M. Koch, D. Kestemont, S. Arangundy-Franklin, and V.B. Pinheiro

5. SpeedyGenesXL An Automated, High-Throughput Platform for the Preparation of Bespoke Ultra-Large Variant Libraries for Directed Evolution
Joanna C. Sadler, Neil Swainston, Mark S. Dunstan, Andrew Currin, and Douglas B. Kell

6. Facile Assembly of Combinatorial Mutagenesis Libraries using Nicking Mutagenesis
Monica B. Kirby and Timothy A. Whitehead

7. GeneORator: An Efficient Method for the Systematic Mutagenesis of Entire Genes
Lucy Green, Nigel S. Scrutton, and Andrew Currin

8. Rapid Cloning of Random Mutagenesis Libraries using PTO-QuickStep
Pawel Jajesniak, Kang Lan Tee, and Tuck Seng Wong

9. Construction of Strong Promoters by Assembling Sigma Factor Binding Motifs
Yonglin Zhang, Yang Wang, Jianghua Li, Chao Wang, Guocheng Du, and Zhen Kang

10. Application of Restriction Free (RF) Cloning in Circular Permutation
Boudhayan Bandyopadhyay and Yoav Peleg

11. Site-Directed Mutagenesis Method Mediated by Cas9
Wanping Chen, Wenwen She, Aitao Li, Chao Zhai, and Lixin Ma

12. Directed Evolution of Transcription Factor-Based Biosensors for Altered Effector Specificity
Leopoldo Ferreira Marques Machado and Neil Dixon

13. A Screening Method for P450 BM3 Mutant Libraries using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds
Anna Gärtner, Gustavo de Almeida Santos, Anna Joëlle Ruff, and Ulrich Schwaneberg

14. Directed Evolution of Glycosyltransferases by a Single-Cell Ultrahigh-Throughput FACS-Based Screening Method
Yumeng Tan, Xue Zhang, Yan Feng, and Guangyu Yang

15. Learning Strategies in Protein Directed Evolution
Xavier F. Cadet, Jean Christophe Gelly, Aster van Noord, Frédéric Cadet, and Carlos G. Acevedo-Rocha

Subject Index List…