Evolutionary methods in biotechnology : clever tricks for directed evolution / edited by Susanne Brakmann and Andreas Schwienhorst.

Format:

Book

Contributor:

Brakmann, Susanne.

Schwienhorst, Andreas.

Language:

English

Subjects (All):

Biotechnology.

Molecular evolution.

Genetic engineering.

Physical Description:

xiii, 214 pages : illustrations ; 25 cm + 1 CD-ROM (4 3/4 in.)

Place of Publication:

Weinheim : Wiley-VCH, [2004]

Summary:

Accompanying CD-ROM contains ... "software tools for library design, selection of mutagenesis positions, and various predictive algorithms." -- p. [4] of cover.

Contents:

2 Generation of Mutant Libraries Using Random Mutagenesis / Susanne Brakmann, Bjorn F. Lindemann 5

2.2 Materials 6

2.2.1 Materials for Random PCR Mutagenesis 6

2.2.2 Materials for Mutator Strain Passage 6

2.3 Protocols 7

2.3.1 Protocol for Random PCR Mutagenesis According to Joyce 7

2.3.2 Protocol for Mutator Strain Passage 8

2.4 Troubleshooting 10

3 DNA Shuffling / Hikaru Suenaga, Masatoshi Goto, Kensuke Furukawa 13

3.2 Materials 15

3.2.1 For Preparation of Parental Genes 15

3.2.2 For Random Fragmentation by DNase I 15

3.2.3 For Collection of DNA Fragments in Specific Molecular Size Ranges 16

3.2.4 For Reassembly of These Fragments by Primerless PCR 16

3.2.5 For Amplification of Reassembled Products by Conventional PCR with Primers 16

3.3 Protocol 17

3.3.1 Preparation of Parental Genes 17

3.3.2 Random Fragmentation by DNase I 17

3.3.3 Collection of DNA Fragments in Specific Molecular Size Ranges 18

3.3.4 Reassembly of These Fragments by Primerless PCR 19

3.3.5 Amplification of Reassembled Products by Conventional PCR with Primers 20

3.4 Troubleshooting 21

3.4.1 Insufficient DNase I Fragmentation 21

3.4.2 Little or No Product of Primerless PCR 21

3.4.3 Little or No Product of PCR with Primers 21

3.4.4 The Product of PCR with Primers is Multi-banded 22

3.5 Amplification Examples 22

4 DNA Recombination Using StEP / Milena Ninkovic 25

4.2 Materials 26

4.2.1 StEP PCR 26

4.2.2 Purification of an Appropriate DNA Fragment 27

4.2.3 Equipment 27

4.3 Protocol 27

4.4 Technical Tips 28

4.4.1 Problem: Little or No PCR Product (Full-length Product) after PCR 28

4.4.2 Problem: High Background Levels of DNA after PCR 28

4.5 StEP in Directed Evolution 29

5 FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells / Thorsten M. Adams, Hans-Ulrich Schmoldt, Harald Kolmar 31

5.2 Materials 35

5.2.1 Escherichia coli Strains and Plasmids 35

5.2.2 Liquid Media and Agar Plates 35

5.2.3 Biological and Chemical Materials 36

5.2.4 Equipment 36

5.3 Protocols 36

5.3.1 Verification of Cell Surface Exposure of the Passenger Protein 36

5.3.2 Labeling of the Target Protein 37

5.3.3 Library Construction 37

5.3.4 Combinatorial Library Screening by FACS and MACS 40

5.4 Troubleshooting 42

5.5 Major Applications 44

6 Selection of Phage-displayed Enzymes / Patrice Soumillion 47

6.2 Materials 50

6.2.1 Buffers, Reagents and Consumables 50

6.2.2 Strains and Vectors 50

6.3 Protocols 51

6.3.1 The Phage-enzyme 51

6.3.2 Library Construction 55

6.3.3 Selection 59

6.4 Troubleshooting 62

6.4.1 Phage Titers Are Not Reproducible 62

6.4.2 Phage-enzymes Degrade with Time 63

6.4.3 Phages Are Not Genetically Stable 63

6.4.4 The 'out/in' Ratio Does Not Increase with Selection Rounds 63

6.5 Major Applications 63

7 Selection of Aptamers / Heiko Fickert, Heike Betat, Ulrich Hahn 65

7.2 Materials 66

7.2.1 Immobilization of Target Molecules 66

7.2.2 PCR 67

7.2.3 In vitro Transcription 67

7.2.4 RNA Purification 67

7.2.5 Selection of Aptamers 67

7.2.6 Reverse Transcription 68

7.3 Protocols 68

7.3.1 Selection of RNA Aptamers 68

7.3.2 Selection of 2'-Modified RNA Aptamers 75

7.3.3 Selection of ssDNA Aptamers 76

7.3.4 Cloning and Sequencing 77

7.3.5 Characterization of Aptamers 77

7.3.6 Example: Isolation of Moenomycin A-specific Aptamers 79

7.4 Troubleshooting 82

7.5 Major Applications 83

8 Methods for Selecting Catalytic Nucleic Acids / Benjamin L. Holley, Bruce E. Eaton 87

8.2 Materials and Equipment 88

8.3 Protocols 91

8.3.1 Generating the Starting Library 91

8.3.2 Transcription 97

8.3.3 Ligation 99

8.3.4 Nucleic Acid-catalyzed Reactions 102

8.3.5 Reverse Transcription 104

8.3.6 Partitioning 105

8.4 Troubleshooting 108

8.5 Major Applications 109

9 High-throughput Screening of Enantioselective Industrial Biocatalysts / Manfred T. Reetz 113

9.2 Materials and Equipment 115

9.2.1 Assays Based on Mass Spectrometry 115

9.2.2 Assays Based on NMR Spectrometry 116

9.2.3 Assay Based on FTIR Spectroscopy 116

9.2.4 Assays Based on UV/Visible Spectroscopy 116

9.2.5 Enzyme-coupled UV/Visible-based Assay for Hydrolases 117

9.3 Protocols 117

9.3.1 Assays Based on Mass Spectrometry 117

9.3.2 Assays Based on NMR Spectroscopy 121

9.3.3 Assay Based on FTIR Spectroscopy 125

9.3.4 Assays Based on UV/Visible Spectroscopy 129

9.3.5 Enzyme-coupled UV/Visible-based Assay for Hydrolases 132

9.3.6 Further Assays 133

9.4 Troubleshooting 138

9.4.1 Comments on the Kazlauskas Test 138

9.4.2 Potential Problems when Performing Kinetic Resolution 138

10 Computer-assisted Design of Doped Libraries / Dirk Tomandl, Andreas Schwienhorst 143

10.2 Materials 146

10.3 Protocol 147

10.4 Troubleshooting 150

10.5 Major Applications 150

11 Directed in silico Mutagenesis / Markus Wiederstein, Peter Lackner, Ferry Kienberger, Manfred J. Sippl 153

11.2 Materials 155

11.2.1 PDB Files 155

11.2.2 Knowledge-based Potentials 156

11.2.3 Polyprotein, Z-scores 160

11.2.4 In silico Mutagenesis 162

11.3 Protocol 163

11.3.1 ProSa Setup and Interaction 163

11.3.2 ProSa Objects 164

11.3.3 Session 1 (mut_script1.cmd) 164

11.3.4 Session 2 (mut_script2.cmd) 166

11.3.5 Session 3 (mut_script3.cmd) 168

11.3.6 Session 4 (mut_script4.cmd) 170

11.3.7 Tips & Tricks 171

11.4 Troubleshooting 173

11.5 Major Applications 174

12 RNA Folding in silico / Christoph Flamm, Ivo L. Hofacker, Peter F. Stadler 177

12.2 Materials 178

12.2.1 Typographical Conventions 179

12.2.2 RNA Web Services 180

12.3 Protocols 181

12.3.1 Secondary Structures for Individual Sequences 181

12.3.2 Consensus Structures of a Sample of Sequences 183

12.3.3 Sequence Design 184

12.3.4 Analysis of SELEX Experiments 186

12.3.5 A Note for the Experts: Write your Own RNA Programs 187

12.4 Troubleshooting 187

12.5 Caveats 188

13 Patenting in Evolutionary Biotechnology / Martina Leimkuhler, Hans-Wilhelm Meyers 191

13.2 The Nature of Patents 191

13.3 What Can Be Patented 192

13.4 The Requirement of Novelty 193

13.5 The Requirement of Inventiveness 196

13.6 The Requirement of Utility 197

13.7 The Requirements of Enablement and Written Description 197

13.8 Patent Prosecution 199

13.9 Search Tools 204

13.10 The First-to-invent Principle of the United States and Its Consequences on Laboratory Notebook Keeping 206.

Notes:

Includes bibliographical references and index.

ISBN:

3527307990

OCLC:

56203779