Evolutionary methods in biotechnology : clever tricks for directed evolution /

1 Introduction Susanne Brakmann and Andreas Schwienhorst p. 1
References p. 3
2 Generation of Mutant Libraries Using Random Mutagenesis Susanne Brakmann and Bjorn F. Lindemann p. 5
2.1 Introduction p. 5
2.2 Materials p. 6
2.2.1 Materials for Random PCR Mutagenesis p. 6
2.2.2 Materials for Mutator Strain Passage p. 6
2.3 Protocols p. 7
2.3.1 Protocol for Random PCR Mutagenesis According to Joyce p. 7
2.3.2 Protocol for Mutator Strain Passage p. 8
2.4 Troubleshooting p. 10
References p. 11
3 DNA Shuffling Hikaru Suenaga and Masatoshi Goto and Kensuke Furukawa p. 13
3.1 Introduction p. 13
3.2 Materials p. 15
3.2.1 For Preparation of Parental Genes p. 15
3.2.2 For Random Fragmentation by DNase I p. 15
3.2.3 For Collection of DNA Fragments in Specific Molecular Size Ranges p. 16
3.2.4 For Reassembly of These Fragments by Primerless PCR p. 16
3.2.5 For Amplification of Reassembled Products by Conventional PCR with Primers p. 16
3.3 Protocol p. 17
3.3.1 Preparation of Parental Genes p. 17
3.3.2 Random Fragmentation by DNase I p. 17
3.3.3 Collection of DNA Fragments in Specific Molecular Size Ranges p. 18
3.3.4 Reassembly of These Fragments by Primerless PCR p. 19
3.3.5 Amplification of Reassembled Products by Conventional PCR with Primers p. 20
3.4 Troubleshooting p. 21
3.4.1 Insufficient DNase I Fragmentation p. 21
3.4.2 Little or No Product of Primerless PCR p. 21
3.4.3 Little or No Product of PCR with Primers p. 21
3.4.4 The Product of PCR with Primers is Multi-banded p. 22
3.5 Amplification Examples p. 22
References p. 23
4 DNA Recombination Using StEP Milena Ninkovic p. 25
4.1 Introduction p. 25
4.2 Materials p. 26
4.2.1 StEP PCR p. 26
4.2.2 Purification of an Appropriate DNA Fragment p. 27
4.2.3 Equipment p. 27
4.3 Protocol p. 27
4.4 Technical Tips p. 28
4.4.1 Problem: Little or No PCR Product (Full-length Product) after PCR p. 28
4.4.2 Problem: High Background Levels of DNA after PCR p. 28
4.5 StEP in Directed Evolution p. 29
References p. 30
5 FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells Thorsten M. Adams and Hans-Ulrich Schmoldt and Harald Kolmar p. 31
5.1 Introduction p. 31
5.2 Materials p. 35
5.2.1 Escherichia coli Strains and Plasmids p. 35
5.2.2 Liquid Media and Agar Plates p. 35
5.2.3 Biological and Chemical Materials p. 36
5.2.4 Equipment p. 36
5.3 Protocols p. 36
5.3.1 Verification of Cell Surface Exposure of the Passenger Protein p. 36
5.3.2 Labeling of the Target Protein p. 37
5.3.3 Library Construction p. 37
5.3.4 Combinatorial Library Screening by FACS and MACS p. 40
5.4 Troubleshooting p. 42
5.5 Major Applications p. 44
References p. 44
6 Selection of Phage-displayed Enzymes Patrice Soumillion p. 47
6.1 Introduction p. 47
6.2 Materials p. 50
6.2.1 Buffers, Reagents and Consumables p. 50
6.2.2 Strains and Vectors p. 50
6.3 Protocols p. 51
6.3.1 The Phage-enzyme p. 51
6.3.2 Library Construction p. 55
6.3.3 Selection p. 59
6.4 Troubleshooting p. 62
6.4.1 Phage Titers Are Not Reproducible p. 62
6.4.2 Phage-enzymes Degrade with Time p. 63
6.4.3 Phages Are Not Genetically Stable p. 63
6.4.4 The 'out/in' Ratio Does Not Increase with Selection Rounds p. 63
6.5 Major Applications p. 63
References p. 64
7 Selection of Aptamers Heiko Fickert and Heike Betat and Ulrich Hahn p. 65
7.1 Introduction p. 65
7.2 Materials p. 66
7.2.1 Immobilization of Target Molecules p. 66
7.2.2 PCR p. 67
7.2.3 In vitro Transcription p. 67
7.2.4 RNA Purification p. 67
7.2.5 Selection of Aptamers p. 67
7.2.6 Reverse Transcription p. 68
7.3 Protocols p. 68
7.3.1 Selection of RNA Aptamers p. 68
7.3.2 Selection of 2'-Modified RNA Aptamers p. 75
7.3.3 Selection of ssDNA Aptamers p. 76
7.3.4 Cloning and Sequencing p. 77
7.3.5 Characterization of Aptamers p. 77
7.3.6 Example: Isolation of Moenomycin A-specific Aptamers p. 79
7.4 Troubleshooting p. 82
7.5 Major Applications p. 83
References p. 83
8 Methods for Selecting Catalytic Nucleic Acids Benjamin L. Holley and Bruce E. Eaton p. 87
8.1 Introduction p. 87
8.2 Materials and Equipment p. 88
8.3 Protocols p. 91
8.3.1 Generating the Starting Library p. 91
8.3.2 Transcription p. 97
8.3.3 Ligation p. 99
8.3.4 Nucleic Acid-catalyzed Reactions p. 102
8.3.5 Reverse Transcription p. 104
8.3.6 Partitioning p. 105
8.4 Troubleshooting p. 108
8.5 Major Applications p. 109
References p. 109
9 High-throughput Screening of Enantioselective Industrial Biocatalysts Manfred T. Reetz p. 113
9.1 Introduction p. 113
9.2 Materials and Equipment p. 115
9.2.1 Assays Based on Mass Spectrometry p. 115
9.2.2 Assays Based on NMR Spectrometry p. 116
9.2.3 Assay Based on FTIR Spectroscopy p. 116
9.2.4 Assays Based on UV/Visible Spectroscopy p. 116
9.2.5 Enzyme-coupled UV/Visible-based Assay for Hydrolases p. 117
9.3 Protocols p. 117
9.3.1 Assays Based on Mass Spectrometry p. 117
9.3.2 Assays Based on NMR Spectroscopy p. 121
9.3.3 Assay Based on FTIR Spectroscopy p. 125
9.3.4 Assays Based on UV/Visible Spectroscopy p. 129
9.3.5 Enzyme-coupled UV/Visible-based Assay for Hydrolases p. 132
9.3.6 Further Assays p. 133
9.4 Troubleshooting p. 138
9.4.1 Comments on the Kazlauskas Test p. 138
9.4.2 Potential Problems when Performing Kinetic Resolution p. 138
9.5 Conclusions p. 139
References p. 139
10 Computer-assisted Design of Doped Libraries Dirk Tomandl and Andreas Schwienhorst p. 143
10.1 Introduction p. 143
10.2 Materials p. 146
10.3 Protocol p. 147
10.4 Troubleshooting p. 150
10.5 Major Applications p. 150
References p. 151
11 Directed in silico Mutagenesis Markus Wiederstein and Peter Lackner and Ferry Kienberger and Manfred J. Sippl p. 153
11.1 Introduction p. 153
11.2 Materials p. 155
11.2.1 PDB Files p. 155
11.2.2 Knowledge-based Potentials p. 156
11.2.3 Polyprotein, Z-scores p. 160
11.2.4 In silico Mutagenesis p. 162
11.2.5 Summary p. 163
11.3 Protocol p. 163
11.3.1 ProSa Setup and Interaction p. 163
11.3.2 ProSa Objects p. 164
11.3.3 Session 1 (mut_script1.cmd) p. 164
11.3.4 Session 2 (mut_script2.cmd) p. 166
11.3.5 Session 3 (mut_script3.cmd) p. 168
11.3.6 Session 4 (mut_script4.cmd) p. 170
11.3.7 Tips & Tricks p. 171
11.4 Troubleshooting p. 173
11.5 Major Applications p. 174
References p. 175
12 RNA Folding in silico Christoph Flamm and Ivo L. Hofacker and Peter F. Stadler p. 177
12.1 Introduction p. 177
12.2 Materials p. 178
12.2.1 Typographical Conventions p. 179
12.2.2 RNA Web Services p. 180
12.3 Protocols p. 181
12.3.1 Secondary Structures for Individual Sequences p. 181
12.3.2 Consensus Structures of a Sample of Sequences p. 183
12.3.3 Sequence Design p. 184
12.3.4 Analysis of SELEX Experiments p. 186
12.3.5 A Note for the Experts: Write your Own RNA Programs p. 187
12.4 Troubleshooting p. 187
12.5 Caveats p. 188
References p. 189
13 Patenting in Evolutionary Biotechnology Martina Leimkuhler and Hans-Wilhelm Meyers p. 191
13.1 Introduction p. 191
13.2 The Nature of Patents p. 191
13.3 What Can Be Patented p. 192
13.4 The Requirement of Novelty p. 193
13.5 The Requirement of Inventiveness p. 196
13.6 The Requirement of Utility p. 197
13.7 The Requirements of Enablement and Written Description p. 197
13.8 Patent Prosecution p. 199
13.9 Search Tools p. 204
13.10 The First-to-invent Principle of the United States and Its Consequences on Laboratory Notebook Keeping p. 206
13.11 Summary p. 209
References p. 209
Subject Index p. 211