Fast and Flexible Synthesis of Combinatorial Libraries for Directed Evolution Joanna C. Sadler, Lucy Green, Neil Swainston, Douglas B. Kell, Andrew Currin 1 School of Chemistry, Faculty of Science and Engineering, Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, University of Manchester, Manchester, United Kingdom 1 Corresponding author: e-mail address: andrew.currin@manchester.ac.uk Contents 1. Introduction 2 1.1 Mutagenesis: Introducing Sequence Variation 4 1.2 Combinatorial Libraries 5 2. Methods 6 2.1 Primer Design 8 2.2 Synthesis of Mutagenic Megaprimers by Asymmetric PCR 9 2.3 Assembly of Full-Length Gene Using Mutagenic Megaprimers 13 3. Library Ligation, Transformation, and Quality Control 14 3.1 Buffers and Reagents 15 3.2 Protocol 15 3.3 Notes and Troubleshooting 15 4. Summary and Conclusion 16 Acknowledgments 17 References 17 Abstract Directed evolution (DE) is a powerful tool for optimizing an enzyme’s properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased k cat . A successful DE project requires the generation of genetic diversity and subsequent screening or selection to identify variants with improved fitness. In contrast to random methods (error-prone PCR or DNA shuffling), site-directed mutagenesis enables the rational design of variant libraries and provides control over the nature and frequency of the encoded mutations. Knowledge of protein structure, dynamics, enzyme mechanisms, and natural evolution demonstrates that multiple Methods in Enzymology # 2018 Elsevier Inc. ISSN 0076-6879 All rights reserved. https://doi.org/10.1016/bs.mie.2018.04.006 1 ARTICLE IN PRESS