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Directed Evolution Library Creation By Elizabeth M.J. Gillam

Directed Evolution Library Creation by Elizabeth M.J. Gillam

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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.

Directed Evolution Library Creation Summary

Directed Evolution Library Creation: Methods and Protocols by Elizabeth M.J. Gillam

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 of Contents

Part I. Point Mutagenesis

(i) Random Mutagenesis Methods

1. Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution

Janine N. Copp, Paulina Hanson-Manful, David F. Ackerley, and Wayne M. Patrick

2. Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesis

Ryota Fujii, Motomitsu Kitaoka, and Kiyoshi Hayashi

3. Random Mutagenesis by Error-Prone Pol Plasmid Replication in Escherichia coli

David L. Alexander, Joshua Lilly, Jaime Hernandez, Jillian Romsdahl, Christopher J. Troll, and Manel Camps

4. The Sequence Saturation Mutagenesis (SeSaM) Method

Anna Joelle Ruff, Tsvetan Kardashliev, Alexander Dennig, and Ulrich Schwaneberg

5. Generation of Effective Libraries by Neutral Drift

Miriam Kaltenbach and Nobuhiko Tokuriki

(ii) Saturation Mutagenesis Methods

6. Site Saturation Mutagenesis

Elsie M. Williams, Janine N. Copp, and David F. Ackerley

7. Iterative Saturation Mutagenesis (ISM): A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution

Carlos G. Acevedo-Rocha, Sabrina Killeand, and Manfred T. Reetz

8. Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides During Gene Shuffling (ISOR)

Liat Rockah-Shmuel, Dan S Tawfik, and Moshe Goldsmith

9. Omni Change: Simultaneous Site-Saturation of up to Five Codons

Alexander Dennig, Jan Marienhagen, Anna Joelle Ruff, and Ulrich Schwaneberg

(iii) Insertional/Deletional Methods

10. Random Insertional-Deletional Strand Exchange Mutagenesis (RAISE): A Simple Method for Generating Random Insertion and Deletion Mutations

Ryota Fujii, Motomitsu Kitaoka, and Kiyoshi Hayashi

11. Transposon-Based Approaches for Generating Novel Molecular Diversity during Directed Evolution

Dafydd Jones, James A.J. Arpino, Amy J. Baldwin, and Matthew C. Edmundson

Part II. Recombinatorial Methods

(i)Homology-Dependent Methods

12. Restriction Enzyme-Mediated DNA Family Shuffling

James B.Y.H. Behrendorff, Wayne A. Johnston, and Elizabeth M. J. Gillam

13. Assembly of Designed Oligonucleotides (ADO): A Useful Tool in Synthetic Biology for Creating High Quality Combinatorial DNA Libraries

Carlos G. Acevedo-Rocha and Manfred T. Reetz

14. One-Pot, Simple Methodology for Cassette Randomization and Recombination for Focused Directed Evolution (OSCARR)

Aurelio Hidalgo Anna Schliessmann, and Uwe T. Bornscheuer

15. USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homology

Benoit Villiers and Florian Hollfelder

(ii) Homology-Independent Methods

16. ITCHY: Incremental Truncation for the Creation of Hybrid Enzymes

Wayne M. Patrick and Monica L. Gerth

17. Generating Random Circular Permutation Libraries

Stefan Lutz, Ashley B. Daugherty, Ying Yu, and Zhen Qian

Part III. Structure Guided Methods and Computational Tools for the Design and Analysis of Mutant Libraries

18. Probabilistic Methods in Directed Evolution: Library Size, Mutation Rate, and Diversity

Yuval Nov

19. The Mutagenesis Assistant Program

Rajni Verma, Tuck Seng Wong, Ulrich Schwaneberg, and Danilo Roccatano

20. Computational Tools for Designing Smart Libraries

Eva Sebestova, Jaroslav Bendl, Jan Brezovsky, and Jiri Damborsky

21. Computational Tools for Directed Evolution: A Comparison of Prospective and Retrospective Strategies

Julian Zaugg, Yosephine Gumulya, Elizabeth M.J. Gillam, and Mikael Boden

22. Designing Libraries of Chimeric Proteins Using SCHEMA Recombination and RASPP

Matthew A. Smith & Frances H. Arnold

23. Non-Contiguous SCHEMA Protein Recombination

Matthew A. Smith and Frances H. Arnold

24. Engineering Proteins by Reconstructing Evolutionary Adaptive Paths

Vanessa E. Cox and Eric A. Gaucher

Additional information

Directed Evolution Library Creation: Methods and Protocols by Elizabeth M.J. Gillam
Springer-Verlag New York Inc.
Book picture is for illustrative purposes only, actual binding, cover or edition may vary.
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