Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering (eBook)

Front Cover 1
Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering 4
Copyright Page 5
Table of Contents 6
Contributors to Volume 421 8
Preface 10
Volumes in Series 12
Section I: Strain Collections and Genetic Nomenclature 36
Chapter 1: Strain Collections and Genetic Nomenclature 38
Introduction 38
Genotype 39
Phenotype 42
References 43
Section II: Transposons 44
Chapter 2: Use of Antibiotic-Resistant Transposons for Mutagenesis 46
Introduction 46
Transposase 47
Delivery of Transposons 48
Transposon Pools 49
References 52
Chapter 3: In Vivo Mutagenesis Using EZ-Tn5TM 52
Introduction 52
Protocol 53
References 56
Chapter 4: Identification of Essential Genes in Bacteria 56
Introduction 56
General Considerations 59
Plan of the Experiment 60
Procedures 63
References 69
Chapter 5: Isolation of Transposon Insertions 70
Introduction 70
References 77
Chapter 6: Localized Mutagenesis 77
Introduction 77
Hydroxylamine Mutagenesis 79
Mutagenesis with Diethylsulfate 82
Hydroxylamine Mutagenesis In Vitro 83
Reagents 84
References 85
Chapter 7: Generation of Deletions and Duplications Using Transposons as Portable Regions of Homology with Emphasis on Mud and Tn10 Transposons 86
Introduction 86
General Considerations 87
References 102
Chapter 8: Target-Directed Proteolysis In Vivo 103
Introduction 104
TEV Protease 104
TEV Protease Expression Vectors 105
Applications of TDP 106
Additional Practical Considerations 114
Additional Potential Applications 115
Acknowledgments 116
References 116
Chapter 9: Sets of Transposon-Generated Sequence-Tagged Mutants for Structure-Function Analysis and Engineering 118
Introduction 118
Permissive Sites 119
Beyond Permissive Sites 119
Flexibility of Insertion Size and Sequence 121
Engineering Permissive Sites with Other Functional Motifs 122
Creation of Derivatives from Permissive i31 Mutants 123
Acknowledgments 124
References 124
Chapter 10: Using Genomic Microarrays to Study Insertional/Transposon Mutant Libraries 125
Introduction 126
Step 1. Generating Insertional Mutant Libraries 128
Step 2. Screening Mutant Libraries: A Primer 128
Step 3. Microarray Analysis of Mutant Pools and Mapping Transposon Insertions 130
Discussion and Conclusions 142
Acknowledgement 144
References 144
Chapter 11: Screening Transposon Mutant Libraries Using Full-Genome Oligonucleotide Microarrays 145
Introduction 145
Principle 147
Transposon Mutagenesis Protocol 150
Library Construction Protocol 152
Competitive Outgrowth of Mutant Libraries Protocol 153
Mutant Labeling Protocol 154
Microarray Design and Hybridization Protocol 156
Data Analysis 157
Conclusion 159
Acknowledgment 160
References 160
Chapter 12: Creating Recombination-Activated Genes and Sequence-Defined Mutant Libraries Using Transposons 161
Introduction 161
Transposon Tn5 Derivatives 161
Recombination-Activated Alleles of Reporter Genes 165
Large-Scale Transposon Mutant Library Construction 167
High-Throughput Mapping of Transposon Insertions 170
Acknowledgments 174
References 174
Chapter 13: Use of Operon and Gene Fusions to Study Gene Regulation in Salmonella 175
Introduction 176
Mud Fusions 176
The Plan of the Experiment 181
Procedures 181
References 193
Chapter 14: Genomic Screening for Regulatory Genes Using the T-POP Transposon 194
Introduction 194
The Plan of the Experiment: The Mutant Hunt 198
References 202
Section III: Phage 204
Chapter 15: Recombineering: In Vivo Genetic Engineering in E. coli, S. enterica, and Beyond 206
Introduction 206
Methods 209
Strains and Plasmids 228
Media 228
Concluding Remarks 230
Acknowledgments 230
References 231
Chapter 16: lambda-Red Genetic Engineering in Salmonella enterica serovar Typhimurium 234
Introduction 235
lambda-Red Mediated Homologous Recombination in S. typhimurium 236
Procedures 238
References 243
Chapter 17: Probing Nucleoid Structure in Bacteria Using Phage Lambda Integrase-Mediated Chromosome Rearrangements 244
Introduction 245
What Is Known About Chromosome Structure 245
Experimental Outline 249
Procedures 250
Comment: Genetic Engineering Using Phage Integrases 258
Conclusion 259
References 259
Chapter 18: Dissecting Nucleic Acid-Protein Interactions Using Challenge Phage 262
Introduction 262
Regulation of Lysis and Lysogeny P22 263
Regulation of ant Gene Expression 265
Selection For and Against DNA Binding in the Challenge Phage System 265
Experimental Procedures 271
Acknowledgments 282
References 282
Chapter 19: Mud-P22 284
Introduction 284
Using Mud-P22 Insertions for Genetic and Physical Analysis of Chromosomal DNA 288
Use of Mud-P22 in Other Species 292
Summary 292
References 292
Chapter 20: Phage Metagenomics 294
Introduction 294
Procedures 296
Acknowledgments 301
References 301
Author Index 304
Subject Index 314