Advanced Bacterial Genetics

FM_II......Page 1
FM_V_VI......Page 2
FM_VII_VIII......Page 4
Preface......Page 6
FM_XI_XXXIV......Page 8
Introduction......Page 32
Insertions......Page 33
Chromosome Rearrangements......Page 35
Conditional Alleles......Page 36
References......Page 37
Introduction......Page 38
Transposase......Page 39
Plasmid Delivery Systems......Page 40
cis Complementation......Page 41
References......Page 44
Introduction......Page 45
Protocol......Page 46
Rescue Cloning......Page 47
References......Page 49
Introduction......Page 50
Construction of Tandem Duplications......Page 53
Isolation of Mud Insertions in and Tn10-araC+ Near Genes of Interest......Page 54
Identification of Essential Genes and Isolation of lac Fusions to Promoters of Essential Genes of Salmonella......Page 55
Materials......Page 57
Materials......Page 58
Materials......Page 59
Materials......Page 61
Materials......Page 62
References......Page 63
Experimental Rationale......Page 64
Protocol 1. Isolation of Mini-Tn10 (Tn10dTet) Insertion Pools......Page 65
Protocol 2. Isolation of Mini-Tn10 (Tn10dTet) Insertions Linked to an Auxotrophic Mutation......Page 66
Testing for Randomness of Transposon Insertions......Page 67
E Medium......Page 69
To Make E + Glucose Plates......Page 70
References......Page 71
Introduction......Page 72
Hydroxylamine Mutagenesis......Page 74
Mutagenesis with Diethylsulfate......Page 77
Hydroxylamine Mutagenesis In Vitro......Page 78
LBSE......Page 79
References......Page 80
Introduction......Page 81
The Transposable Elements, Tn10 MudA and MudB......Page 82
Generation of Deletions and Duplications Using Tn10 or Mud by P22 Transduction Tn10......Page 83
Mud......Page 85
Mud......Page 87
Tn10......Page 89
Materials......Page 90
Materials......Page 92
Materials......Page 94
Materials......Page 96
References......Page 97
Target-Directed Proteolysis In Vivo......Page 99
TEV Protease......Page 100
TEV Protease Expression Vectors......Page 101
Inactivation of Essential Proteins in the Cytoplasm......Page 102
Proteolysis at the Ribosome......Page 105
Structure-Function Studies of Integral Outer Membrane Proteins......Page 107
TCA Precipitation......Page 108
Purification of Insoluble (His)6-TEV Protease......Page 109
Additional Practical Considerations......Page 110
Additional Potential Applications......Page 111
References......Page 112
Introduction......Page 115
Beyond Permissive Sites......Page 116
Flexibility of Insertion Size and Sequence......Page 118
Engineering Permissive Sites with Other Functional Motifs......Page 119
Creation of Derivatives from Permissive i31 Mutants......Page 120
References......Page 121
Using Genomic Microarrays to Study Insertional/Transposon Mutant Libraries......Page 123
Introduction......Page 124
Step 2. Screening Mutant Libraries: A Primer......Page 126
Basic Experimental Flow......Page 128
Preparation of Bacterial Genomic DNA......Page 129
Amplification of Transposon-Containing DNA: First-Round PCR......Page 130
Reamplification and Incorporation of aa-dUTP into Transposon-Flanking DNA: Second-Round PCR......Page 131
Purification and Labeling of Second-Round PCR Products......Page 132
Purification and Preparation of Labeled PCR Product for Hybridization......Page 133
Hybridization and Washing Spotted Microarrays......Page 134
Data Collection and Normalization......Page 135
Data Storage......Page 136
Data Analysis: Screening for Minimally Essential Genes......Page 137
Data Analysis: Screening for Conditionally Essential Genes Such as Colonization Factors......Page 138
Discussion and Conclusions......Page 140
References......Page 142
Introduction......Page 144
Principle......Page 146
Tn5 Transposome Complexes......Page 149
In Vivo Tn5 Mutagenesis......Page 150
Library Construction Protocol......Page 151
Competitive Outgrowth of Mutant Libraries Protocol......Page 152
DNA Template Preparation......Page 153
In Vitro Transcription Reaction......Page 154
Microarray Design and Hybridization Protocol......Page 155
Data Analysis......Page 156
Conclusion......Page 158
References......Page 159
Transposon Tn5 Derivatives......Page 160
Delivery......Page 161
Identifying Cre-Activated lacZ Derivatives Using ISOmega/hah......Page 164
Generating and Arraying Mutants......Page 166
High-Throughput Mapping of Transposon Insertions......Page 169
References......Page 173
Use of Operon and Gene Fusions to Study Gene Regulation in Salmonella......Page 175
The Transposable Elements, MudJ and MudK......Page 176
Use of Phage P22 to Transduce Mud......Page 179
Diagnosis of Auxotrophs (Auxanography)......Page 180
Strains......Page 181
Materials......Page 182
Discussion......Page 184
Introduction: The Flagellum......Page 186
Isolation of MudJ/K Insertions in Genes Required for Flagella Assembly......Page 187
The Plan of the Experiment: The Mutant Hunt......Page 188
Preparation......Page 189
Introduction......Page 191
Transposition of TnphoA......Page 192
References......Page 193
The Transposable Element, T-POP......Page 194
Use of Phage P22 to Transduce T-POP......Page 196
Use of T-POP to Identify Genes Affecting Regulation of Gene Expression......Page 197
The Plan of the Experiment: The Mutant Hunt......Page 198
MudP22 Lysate Preparation......Page 199
Materials......Page 200
Procedure 3. Arbitrary PCR for Sequencing Out of the T-POP Mutants......Page 201
References......Page 202
What Is Recombineering?......Page 203
Red Proteins and Properties......Page 204
Expression of Red Proteins from a Defective Prophage......Page 205
Standard Recombineering Protocol......Page 206
Transformation by Electroporation......Page 207
Outgrowth......Page 208
Selection or Screening for Mutants......Page 209
Confirming Mutations......Page 210
Elimination of the lambda Stuff......Page 211
Preparing Linear dsDNA......Page 213
Methyl-Directed Mismatch Repair Mutants......Page 214
Other Means of Maximizing Recombination......Page 216
Selection/Counter-Selection for Gene Mutation, Replacement, and Fusion......Page 217
Duplications......Page 218
Gene-Specific Random Mutagenesis Using Recombineering......Page 219
Recombineering on Plasmids......Page 220
Gap Repair of Plasmids: In Vivo Cloning......Page 221
Targeting Recombineering to Phage: Modifications to the Standard Protocol......Page 223
Prophage-Containing Recombineering Plasmids......Page 224
M63-DOG (for Selecting GalK Mutants)......Page 225
Acknowledgments......Page 227
References......Page 228
lambda-Red Genetic Engineering in Salmonella enterica serovar Typhimurium......Page 232
Introduction......Page 233
lambda-Red Mediated Homologous Recombination in S. typhimurium......Page 234
Reagents......Page 236
Primer Design for rrnB T1T2 tetAR2......Page 237
Day 2: Preparation of Cells for lambda-Red-Mediated Recombination......Page 238
Primer Designs for In-Frame Deletions, Gene Fusions, and Site-Directed Mutagenesis......Page 239
Days 3 and 4......Page 240
References......Page 241
Probing Nucleoid Structure in Bacteria Using Phage Lambda Integrase-Mediated Chromosome Rearrangements......Page 243
What Is Known About Chromosome Structure......Page 244
Experimental Outline......Page 248
Preparation......Page 249
Procedure 2. qPCR Measurement of Chromosome Fluidity......Page 252
Strains......Page 253
Preparation......Page 254
Comment: Genetic Engineering Using Phage Integrases......Page 257
References......Page 258
Introduction......Page 261
Regulation of Lysis and Lysogeny P22......Page 262
Selection For and Against DNA Binding in the Challenge Phage System......Page 264
Construction of Challenge Phage with Novel Operator Sequences at Pant......Page 265
Bacteria and Phage......Page 270
Standard High-Titer P22 Lysate Preparation from Single Plaques......Page 271
Construction of Challenge Phage by In Vivo Recombination......Page 273
PCR and RFLP Analysis......Page 274
Procedure 4. Challenge Phage DNA-Binding Assays......Page 275
Challenge Phage Assays......Page 276
Procedure 5. Selection for DNA-Binding Site Mutations......Page 277
Isolation of Mutations in a DNA-Binding Site......Page 278
Purify Clear Plaque Mutants......Page 279
Procedure 6. Construction of Challenge Phages Requiring Multiple Binding Sites......Page 280
References......Page 281
Phage Mu......Page 284
Mud-P22......Page 285
Isolation of Chromosomal Mud-P22 Insertions......Page 286
Induction of Mud-P22 Lysogens......Page 287
Using Mud-P22 Insertions for Genetic and Physical Analysis of Chromosomal DNA......Page 288
Rapid Genetic Mapping......Page 290
DNA Hybridization......Page 291
References......Page 292
Introduction......Page 295
Protocol 1. Isolation of Viral Particles from Soil and Sediment Environmental Samples......Page 297
Protocol 2. Isolation of Viral Particles from Large Water Environmental Samples Using Tangential-Flow Filtration......Page 298
Protocol 3. Purification of Viral Particles by Cesium Chloride (CsCl) Density Centrifugation......Page 299
Formamide Preparation......Page 300
CTAB/NaCl Preparation......Page 301
References......Page 302
21......Page 305
22......Page 315