Prokaryotic GenomicsMichel 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
1 | |
Methods | 2 |
Preparation of bacterial DNA | 3 |
Resolving multiple fragments following electrophoresis | 5 |
Results and discussion | 6 |
References | 9 |
10 | |
Genetic mapping in Salmonella | 11 |
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 |
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 |
34 | |
Methods | 36 |
DNA sample preparation using Chelex Sigma TM | 37 |
Length analysis | 38 |
Troubleshooting | 39 |
Acknowledgments | 40 |
41 | |
Materials and methods | 42 |
Protocols | 43 |
Transfer of duplications from strain to strain | 46 |
Results and discussion | 47 |
Concluding remarks | 48 |
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 |
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 |
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 |
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 |
131 | |
Materials | 135 |
Scanner | 136 |
cDNA synthesis and labeling | 137 |
Troubleshooting | 138 |
Webbased microarray resources | 140 |
Commercial arrays | 141 |
References | 142 |
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 |
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 |
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 |
191 | |
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 |
205 | |
207 | |
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Häufige Begriffe und Wortgruppen
Acad Sci USA acid acyl-HSL agar analysis antibiotic arrays assay bacterial Bacteriol bacteriophage bioluminescence biosensor broth buffer Buffer Solution carboxy-terminal cell Centrifuge chromosomal cleavage cloning containing copy number cotransduction CRIM plasmids culture detection digestion dilution DNA fragments DNA microarray duplication electrophoresis Escherichia coli evolution extract fusion vectors gene expression genetic footprinting genome growth homologous recombination host hybridization Incubate induction insertion integration intein IPTG isolated lactone lacZ leuA414 r m ligation lysate lysogens mapping marker methods microarray Microbiol microfuge microplate microsatellites ml LB molecular mutations Natl Acad Sci operon PCR products pellet PFGE phenotypic plate populations primers Proc Natl Acad prophages Protocol purification quorum sensing r m fels2 recombination residue restriction enzyme resuspended Salmonella samples segregants selection sequence signal solution ẞ-galactosidase strain supernatant T-DNA target gene target protein temperature tion transcriptional transcriptome transducing phage transduction transfer transposon tube typhimurium X-gal µg/ml