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

Description

The critically acclaimed laboratory standard for more than fifty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 400 volumes (all of them still in print), the series contains much material still relevant today—truly an essential publication for researchers in all fields of life sciences. This new volume presents methods related to the use of bacterial genetics for genomic engineering. The book includes sections on strain collections and genetic nomenclature; transposons; and phage.

Front Cover 2
Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering 5
Copyright Page 6
Table of Contents 7
Contributors to Volume 421 9
Preface 11
Volumes in Series 13
Section I: Strain Collections and Genetic Nomenclature 37
- Chapter 1: Strain Collections and Genetic Nomenclature 39
  - Introduction 39
  - Genotype 40
  - Phenotype 43
  - References 44
- Section II: Transposons 45

Front Cover 2
Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering 5
Copyright Page 6
Table of Contents 7
Contributors to Volume 421 9
Preface 11
Volumes in Series 13
Section I: Strain Collections and Genetic Nomenclature 37
- Chapter 1: Strain Collections and Genetic Nomenclature 39
  - Introduction 39
  - Genotype 40
  - Phenotype 43
  - References 44
- Section II: Transposons 45
  - Chapter 2: Use of Antibiotic-Resistant Transposons for Mutagenesis 47
    - Introduction 47
    - Transposase 48
    - Delivery of Transposons 49
    - Transposon Pools 50
    - References 53
  - Chapter 3: In Vivo Mutagenesis Using EZ-Tn5TM 53
    - Introduction 53
    - Protocol 54
    - References 57
  - Chapter 4: Identification of Essential Genes in Bacteria 57
    - Introduction 57
    - General Considerations 60
    - Plan of the Experiment 61
    - Procedures 64
    - References 70
  - Chapter 5: Isolation of Transposon Insertions 71
    - Introduction 71
    - References 78
  - Chapter 6: Localized Mutagenesis 78
    - Introduction 78
    - Hydroxylamine Mutagenesis 80
    - Mutagenesis with Diethylsulfate 83
    - Hydroxylamine Mutagenesis In Vitro 84
    - Reagents 85
    - References 86
  - Chapter 7: Generation of Deletions and Duplications Using Transposons as Portable Regions of Homology with Emphasis on Mud and T 87
    - Introduction 87
    - General Considerations 88
    - References 103
  - Chapter 8: Target-Directed Proteolysis In Vivo 104
    - TEV Protease 105
    - Introduction 105
    - TEV Protease Expression Vectors 106
    - Applications of TDP 107
    - Additional Practical Considerations 115
    - Additional Potential Applications 116
    - References 117
    - Acknowledgments 117
  - Chapter 9: Sets of Transposon-Generated Sequence-Tagged Mutants for Structure-Function Analysis and Engineering 119
    - Introduction 119
    - Permissive Sites 120
    - Beyond Permissive Sites 120
    - Flexibility of Insertion Size and Sequence 122
    - Engineering Permissive Sites with Other Functional Motifs 123
    - Creation of Derivatives from Permissive i31 Mutants 124
    - Acknowledgments 125
    - References 125
  - Chapter 10: Using Genomic Microarrays to Study Insertional/Transposon Mutant Libraries 126
    - Introduction 127
    - Step 2. Screening Mutant Libraries: A Primer 129
    - Step 1. Generating Insertional Mutant Libraries 129
    - Step 3. Microarray Analysis of Mutant Pools and Mapping Transposon Insertions 131
    - Discussion and Conclusions 143
    - Acknowledgement 145
    - References 145
  - Chapter 11: Screening Transposon Mutant Libraries Using Full-Genome Oligonucleotide Microarrays 146
    - Introduction 146
    - Principle 148
    - Transposon Mutagenesis Protocol 151
    - Library Construction Protocol 153
    - Competitive Outgrowth of Mutant Libraries Protocol 154
    - Mutant Labeling Protocol 155
    - Microarray Design and Hybridization Protocol 157
    - Data Analysis 158
    - Conclusion 160
    - References 161
    - Acknowledgment 161
  - Chapter 12: Creating Recombination-Activated Genes and Sequence-Defined Mutant Libraries Using Transposons 162
    - Transposon Tn5 Derivatives 162
    - Introduction 162
    - Recombination-Activated Alleles of Reporter Genes 166
    - Large-Scale Transposon Mutant Library Construction 168
    - High-Throughput Mapping of Transposon Insertions 171
    - References 175
    - Acknowledgments 175
  - Chapter 13: Use of Operon and Gene Fusions to Study Gene Regulation in Salmonella 176
    - Mud Fusions 177
    - Introduction 177
    - Procedures 182
    - The Plan of the Experiment 182
    - References 194
  - Chapter 14: Genomic Screening for Regulatory Genes Using the T-POP Transposon 195
    - Introduction 195
    - The Plan of the Experiment: The Mutant Hunt 199
    - References 203
  - Section III: Phage 205
    - Chapter 15: Recombineering: In Vivo Genetic Engineering in E. coli, S. enterica, and Beyond 207
      - Introduction 207
      - Methods 210
      - Media 229
      - Strains and Plasmids 229
      - Concluding Remarks 231
      - Acknowledgments 231
      - References 232
    - Chapter 16: lambda-Red Genetic Engineering in Salmonella enterica serovar Typhimurium 235
      - Introduction 236
      - lambda-Red Mediated Homologous Recombination in S. typhimurium 237
      - Procedures 239
      - References 244
    - Chapter 17: Probing Nucleoid Structure in Bacteria Using Phage Lambda Integrase-Mediated Chromosome Rearrangements 245
      - What Is Known About Chromosome Structure 246
      - Introduction 246
      - Experimental Outline 250
      - Procedures 251
      - Comment: Genetic Engineering Using Phage Integrases 259
      - References 260
      - Conclusion 260
    - Chapter 18: Dissecting Nucleic Acid-Protein Interactions Using Challenge Phage 263
      - Introduction 263
      - Regulation of Lysis and Lysogeny P22 264
      - Selection For and Against DNA Binding in the Challenge Phage System 266
      - Regulation of ant Gene Expression 266
      - Experimental Procedures 272
      - References 283
      - Acknowledgments 283
    - Chapter 19: Mud-P22 285
      - Introduction 285
      - Using Mud-P22 Insertions for Genetic and Physical Analysis of Chromosomal DNA 289
      - Summary 293
      - References 293
      - Use of Mud-P22 in Other Species 293
    - Chapter 20: Phage Metagenomics 295
      - Introduction 295
      - Procedures 297
      - References 302
      - Acknowledgments 302
    - Author Index 305
    - Subject Index 315

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