Prokaryotic Genomics

Cover
Michel Blot
Springer Science & Business Media, 11.12.2002 - 208 Seiten
This manual reflects practical approaches to handling bacteria in the labora- tory. It is designed to recall historical methods of bacterial genetics that have had recent developments and to present new techniques that allow full genome analysis. It has been written for microbiologists who need to group their protocols at the state of the art of a new millennium and also for scientists in other fields of life sciences who need to use bacteria for their research. Teachers, graduate students, and postdocs also will benefit from having these protocols to help them understand modern bacterial genetics. I learned so much from these contributions from my colleagues that I have no doubt about the daily usefulness of this book. April 2002 Michel Blot XII Abbreviations Acyl-HSL N-acyl homoserine lactone moi multiplicity of infection Amp or Ap ampicillin N amino C carboxy NMR nuclear magnetic resonance CIO-HSL N-decanoyl-L-homoserine lactone 3-0H-C14:1-HSL N-(3-hydroxy-7 -cis-tetra- C12-HSL N-dodecanoyl-L-homoserine lac- decanoyl)homo-serine lactone tone 3-0H-C4-HSL N-3-hydroxybutanoyl-L- C14-HSL N-tetradecanoyl-L-homoserine homoserine lactone lactone ONPG o-nitrophenyl -D-galactopyranoside C4-HSL N-butanoyl-L-homoserine lactone ORF open reading frame C6-HSL N-hexanoyl-L-homoserine lactone OTG I-S-octyl- -D-thioglucoside C8-HSL N-octanoyl-L-homoserine lactone 3-oxo-CIO-HSL N-3-oxodecanoyl-L-homo- Cam or Cm chloramphenicol serine lactone CBD chitin binding domain 3-oxo-C12-HSL N-3-oxododecanoyl-L- CHEF contour clamped homogenous electric homoserine lactone field 3-oxo-C14-HSL N-3-oxotetradecanoyl-L- CI consistency index homoserine lactone CRIM conditional-replication, integration, 3-oxo-C4-HSL N-3-oxobutanoyl-L-homoser- and modular ine lactone dCTP deoxycytidine triphosphate 3-oxo-C6-HSL N-3 -oxohexanoyl-L-homoser- deg.
 

Inhalt

Physical Analysis of Chromosome Size Variation
1
Methods
2
Preparation of bacterial DNA
3
Resolving multiple fragments following electrophoresis
5
Results and discussion
6
References
9
Genetic Mapping in Salmonella enterica
10
Genetic mapping in Salmonella
11
Quorum Sensing Approaches to Identify Signals and Signalling Genes in Gramnegative Bacteria
110
Introduction
111
Materials
112
Equipment
113
Methods
114
Reporter strains
115
Solvent extraction of signal molecules
120
TLC assay
121

Genetic mapping by duplication segregation
14
Materials
16
Methods
17
Mapping
18
Troubleshooting
19
Acknowledgments
20
Insertion Sequences as Genomic Markers
22
Materials
24
DNA digestion transfer onto nylon membranes and hybridization experiments
25
Results and discussion
26
10000 generations of experimental evolution in E coli
27
Mapping the pivotal ISlinked mutations
28
IS mutations in other experimental evolution populations
30
Acknowledgments
31
The Use of Noncoding Microsatellite Length Analysis for Bacterial Strain Typing
34
Methods
36
DNA sample preparation using Chelex Sigma TM
37
Length analysis
38
Troubleshooting
39
Acknowledgments
40
How to Amplify Easily on the Bacterial Chromosome a Desired DNA Sequence
41
Materials and methods
42
Protocols
43
Transfer of duplications from strain to strain
46
Results and discussion
47
Concluding remarks
48
Generalized Transduction
50
Materials
51
Methods
52
Checking Salmonella for P22 sensitivity
54
Transduction with Plvir
55
Results and discussion
57
Generalized transduction by phage PI
58
Inheritance of chromosomal DNA via homologous recombination
59
Transduction of plasmids
61
Acknowledgments
62
Use of Conditionalreplication Integration and Modular CRIM Plasmids to Make Singlecopy lacZ Fusions
65
Materials
69
Nucleotide sequence accession numbers
71
Equipment
72
Methods
73
Preparation of electrocompetent cells
74
PCR test of integrant copy number
75
Simple plate test for estimation of pgalactosidase activity
77
Troubleshooting
80
Applications
81
Remarks and conclusions
85
Acknowledgements
86
References
87
Genetic Footprinting for Bacterial Functional Genomics
90
Materials
92
Solutions
93
Equipment and software
95
Outgrowth of mutagenized cell populations Isolation of the T15 T30 and T45 populations
96
Polymerase chain reaction
97
Applications
98
Remarks and conclusions
99
Acknowledgments
100
Gene Transfer to Plants through Bacterial Vectors
102
Materials
104
Methods
105
Histochemical XGlu assay
106
Applications
107
Acknowledgments
109
Gene cloning
122
The QS regulon
123
What is the phenotype of the null mutants?
124
Applications
126
Transposon mutagenesis to identify phenotypic characteristics
127
Acknowledgements
128
Transcriptional Profiling in Bacteria Using Microarrays
131
Materials
135
Scanner
136
cDNA synthesis and labeling
137
Troubleshooting
138
Webbased microarray resources
140
Commercial arrays
141
References
142
Transcriptome Analysis by Macroarrays
145
Materials
148
Preparation of the bacterial cells
149
RNA precipitation
150
cDNA synthesis
151
Hybridization
152
Exposure to phosphorimager screens
153
Applications
154
Remarks and conclusions
155
Further reading
156
Prokaryotic Proteomics
157
Introduction
158
Materials
159
Staining solutions
160
Methods
161
French press
162
Synechocystis 6803
163
Rehydration step
164
Prepare IPG strip for isofocalization
165
Second dimension
166
Silver staining
167
Protein characterization
168
Troubleshooting
171
Inteinmediated Protein Purification
172
Introduction
173
Materials
175
E coli strains
176
Methods
177
Cloning a target gene into an intein vector
180
Primer design
181
Amplification of a target gone
182
Screening for target gene inserts
183
Induction
184
Loading clarified cell extract on a chitin column
185
Elution of the target protein
186
Troubleshooting
187
Applications
188
Remarks and conclusions
191
Twohybrid Assay in Escherichia coli K12
194
Materials and equipment
196
Media solutions and buffers
197
Cloning the genes of interest
198
Cloning
199
Assembling of the twohybrid system
200
Quantification of proteinprotein interaction
201
Troubleshooting
202
References
203
Guide to Protocols
205
Index
207
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