Evolutionary Methods in Biotechnology
Clever Tricks for Directed Evolution
with CD-ROM
edited by Susanne Brakmann

From library generation by random or directed mutagenesis to screening and selection techniques - the crucial steps for successful evolutionary biotechnology are described in detail in this practical guide that also includes valuable troubleshooting hints on frequently encountered problems.

Modern methods for the surface display of peptides and proteins, selective enrichment of nucleic acid aptamers and high-throughput screening of industrial biocatalysts are explained, and computer-based methods for in silico protein and RNA engineering are described as an alternative to in vitro approaches. A special section covers the patenting regulations with regard to biotechnological innovations derived from directed evolution.

As an added bonus, a CD-ROM is included that contains software tools for library design, selection of mutagenesis positions, and various predictive algorithms.

In short, this practice oriented handbook is an indispensable tool for every scientist working in this interdisciplinary research area.

Contents

1. Introduction

2. Generation of Mutant Libraries Using Random Mutagenesis
   • Introduction.
   • Materials.
   • Protocols.
   • Troubleshooting.

3. DNA Shuffling
   • Introduction.
   • Materials.
   • Protocol.
   • Troubleshooting.
   • Amplification Examples.22

4. DNA Recombination Using StEP
   • Introduction.
   • Materials.
   • Protocol.
   • Technical Tips.
   • StEP in Directed Evolution.

5. FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells
   • Introduction.
   • Materials.
   • Protocols.
   • Troubleshooting.
   • Major Applications.

6. Selection of Phage-displayed Enzymes
   • Introduction.
   • Materials.
   • Protocols.
   • Troubleshooting.
   • Major Applications.

7. Selection of Aptamers
   • Introduction.
   • Materials.
   • Protocols.
   • Troubleshooting.
   • Major Applications.

8. Methods for Selecting Catalytic Nucleic Acids
   • Introduction.
   • Materials and Equipment.
   • Protocols.
   • Troubleshooting.
   • Major Applications.

9. High-throughput Screening of Enantioselective Industrial Biocatalysts
   • Introduction.
   • Materials and Equipment.
   • Protocols.
   • Troubleshooting.
   • Conclusions

10. Computer-assisted Design of Doped Libraries
    • Introduction.
    • Materials.
    • Protocol.
    • Troubleshooting.
    • Major Applications.

11. Directed in silico Mutagenesis
    • Introduction.
    • Materials.
    • Protocol.
    • Troubleshooting.
    • Major Applications

12. RNA Folding in silico
    • Introduction.
    • Materials.
    • Protocols.
    • Troubleshooting.
    • Caveats.

13. Patenting in Evolutionary Biotechnology
    • Introduction.
    • The Nature of Patents.
    • What Can Be Patented.
    • The Requirement of Novelty.
    • The Requirement of Inventiveness.
    • The Requirement of Utility.
    • The Requirements of Enablement and Written Description.
    • Patent Prosecution.
    • Search Tools.
    • The First-to-invent Principle of the United States and Its Consequences on Laboratory Notebook Keeping

Subject Index

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