Directed Evolution: Methods and Protocols

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 Grtner, Gustavo de Almeida Santos, Anna Jolle 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, Frdric Cadet, and Carlos G. Acevedo-Rocha

Subject Index List...