References.

2 Generation of Mutant Libraries Using Random Mutagenesis (Susanne Brakmann and Björn F. Lindemann).

2.1 Introduction.

2.2 Materials.

2.2.1 Materials for Random PCR Mutagenesis.

2.2.2 Materials for Mutator Strain Passage.

2.3 Protocols.

2.3.1 Protocol for Random PCR Mutagenesis According to Joyce.

2.3.2 Protocol for Mutator Strain Passage.

2.4 Troubleshooting.

References.

3 DNA Shuffling (Hikaru Suenaga, Masatoshi Goto, and Kensuke Furukawa).

3.1 Introduction.

3.2 Materials.

3.2.1 For Preparation of Parental Genes.

3.2.2 For Random Fragmentation by DNase I.

3.2.3 For Collection of DNA Fragments in Specific Molecular Size Ranges.

3.2.4 For Reassembly of These Fragments by Primerless PCR.

3.2.5 For Amplification of Reassembled Products by Conventional PCR with Primers.

3.3 Protocol.

3.3.1 Preparation of Parental Genes.

3.3.2 Random Fragmentation by DNase I.

3.3.3 Collection of DNA Fragments in Specific Molecular Size Ranges.

3.3.4 Reassembly of These Fragments by Primerless PCR.

3.3.5 Amplification of Reassembled Products by Conventional PCR with Primers.

3.4 Troubleshooting.

3.4.1 Insufficient DNase I Fragmentation.

3.4.2 Little or No Product of Primerless PCR.

3.4.3 Little or No Product of PCR with Primers.

3.4.4 The Product of PCR with Primers is Multi-banded.

3.5 Amplification Examples.22

References.

4 DNA Recombination Using StEP (Milena Ninkovic).

4.1 Introduction.

4.2 Materials.

4.2.1 StEP PCR.

4.2.2 Purification of an Appropriate DNA Fragment.

4.2.3 Equipment.

4.3 Protocol.

4.4 Technical Tips.

4.4.1 Problem: Little or No PCR Product (Full-length Product) after PCR.

4.4.2 Problem: High Background Levels of DNA after PCR.

4.5 StEP in Directed Evolution.

References.

5 FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells (Thorsten M. Adams, Hans-Ulrich Schmoldt, and Harald Kolmar).

5.1 Introduction.

5.2 Materials.

5.2.1 Escherichia coli Strains and Plasmids.

5.2.2 Liquid Media and Agar Plates.

5.2.3 Biological and Chemical Materials.

5.2.4 Equipment.

5.3 Protocols.

5.3.1 Verification of Cell Surface Exposure of the Passenger Protein.

5.3.2 Labeling of the Target Protein.

5.3.3 Library Construction.

5.3.4 Combinatorial Library Screening by FACS and MACS.

5.4 Troubleshooting.

5.5 Major Applications.

References.

6 Selection of Phage-displayed Enzymes (Patrice Soumillion).

6.1 Introduction.

6.2 Materials.

6.2.1 Buffers, Reagents and Consumables.

6.2.2 Strains and Vectors.

6.3 Protocols.

6.3.1 The Phage-enzyme.

6.3.2 Library Construction.

6.3.3 Selection.

6.4 Troubleshooting.

6.4.1 Phage Titers Are Not Reproducible.

6.4.2 Phage-enzymes Degrade with Time.

6.4.3 Phages Are Not Genetically Stable.

6.4.4 The ‘out/in’ Ratio Does Not Increase with Selection Rounds.

6.5 Major Applications.

References.

7 Selection of Aptamers (Heiko Fickert, Heike Betat, and Ulrich Hahn).

7.1 Introduction.

7.2 Materials.

7.2.1 Immobilization of Target Molecules.

7.2.2 PCR.

7.2.3 In vitro Transcription.

7.2.4 RNA Purification.

7.2.5 Selection of Aptamers.

7.2.6 Reverse Transcription.

7.3 Protocols.

7.3.1 Selection of RNA Aptamers.

7.3.2 Selection of 2_-Modified RNA Aptamers.

7.3.3 Selection of ssDNA Aptamers.

7.3.4 Cloning and Sequencing.

7.3.5 Characterization of Aptamers.

7.3.6 Example: Isolation of Moenomycin A-specific Aptamers.

7.4 Troubleshooting.

7.5 Major Applications.

References.

8 Methods for Selecting Catalytic Nucleic Acids (Benjamin L. Holley, and Bruce E. Eaton).

8.1 Introduction.

8.2 Materials and Equipment.

8.3 Protocols.

8.3.1 Generating the Starting Library.

8.3.2 Transcription.

8.3.3 Ligation.

8.3.4 Nucleic Acid-catalyzed Reactions.

8.3.5 Reverse Transcription.

8.3.6 Partitioning.

8.4 Troubleshooting.

8.5 Major Applications.

References.

9 High-throughput Screening of Enantioselective Industrial Biocatalysts (Manfred T. Reetz).

9.1 Introduction.

9.2 Materials and Equipment.

9.2.1 Assays Based on Mass Spectrometry.

9.2.2 Assays Based on NMR Spectrometry.

9.2.3 Assay Based on FTIR Spectroscopy.

9.2.4 Assays Based on UV/Visible Spectroscopy.

9.2.5 Enzyme-coupled UV/Visible-based Assay for Hydrolases.

9.3 Protocols.

9.3.1 Assays Based on Mass Spectrometry.

9.3.2 Assays Based on NMR Spectroscopy.

9.3.3 Assay Based on FTIR Spectroscopy.

9.3.4 Assays Based on UV/Visible Spectroscopy.

9.3.5 Enzyme-coupled UV/Visible-based Assay for Hydrolases.

9.3.6 Further Assays.

9.4 Troubleshooting.

9.4.1 Comments on the Kazlauskas Test.

9.4.2 Potential Problems when Performing Kinetic Resolution.

9.5 Conclusions.

References.

10 Computer-assisted Design of Doped Libraries (Dirk Tomandl and Andreas Schwienhorst).

10.1 Introduction.

10.2 Materials.

10.3 Protocol.

10.4 Troubleshooting.

10.5 Major Applications.

References.

11 Directed in silico Mutagenesis (Markus Wiederstein, Peter Lackner, Ferry Kienberger, Manfred J. Sippl).

11.1 Introduction.

11.2 Materials.

11.2.1 PDB Files.

11.2.2 Knowledge-based Potentials.

11.2.3 Polyprotein, Z-scores.

11.2.4 In silico Mutagenesis.

11.2.5 Summary.

11.3 Protocol.

11.3.1 ProSa Setup and Interaction.

11.3.2 ProSa Objects.

11.3.3 Session 1 (mut script1.cmd).

11.3.4 Session 2 (mut script2.cmd).

11.3.5 Session 3 (mut script3.cmd).

11.3.6 Session 4 (mut script4.cmd).

11.3.7 Tips & Tricks.

11.4 Troubleshooting.

11.5 Major Applications.

References.

12 RNA Folding in silico(Christoph Flamm, Ivo L. Hofacker, Peter F. Stadler).

12.1 Introduction.

12.2 Materials.

12.2.1 Typographical Conventions.

12.2.2 RNA Web Services.

12.3 Protocols.

12.3.1 Secondary Structures for Individual Sequences.

12.3.2 Consensus Structures of a Sample of Sequences.

12.3.3 Sequence Design.

12.3.4 Analysis of SELEX Experiments.

12.3.5 A Note for the Experts: Write your Own RNA Programs.

12.4 Troubleshooting.

12.5 Caveats.

References.

13 Patenting in Evolutionary Biotechnology (Martina Leimkühler and Hans-Wilhelm Meyers).

13.1 Introduction.

13.2 The Nature of Patents.

13.3 What Can Be Patented.

13.4 The Requirement of Novelty.

13.5 The Requirement of Inventiveness.

13.6 The Requirement of Utility.

13.7 The Requirements of Enablement and Written Description.

13.8 Patent Prosecution.

13.9 Search Tools.

13.10 The First-to-invent Principle of the United States and Its Consequences on Laboratory Notebook Keeping.

13.11 Summary.

References.

Subject Index.