Coordinated regulation of gene expression by E ... - Jacobs University

More documents

Recommendations

Info

MATERIALS AND METHODS 2.2.10 Growth curves and norfloxacin treatment The growth curves for CSH50 and LZ strains were generated from two overnight cultures; the first one was in LB and the second in 2X YT medium. The second overnight culture was diluted 1:200 times in appropriate volume of 2X YT and incubated at 37 o C in the case of CSH50 strains and at 30 o C for LZ strains, with shaking at 150rpm. The optical density measurements (OD) were recorded at 600nm wavelength (using Eppendorf ® Biophotometer). Sampling of cultures was carried out by pipetting the required volume of culture into frozen falcon tubes pre-filled with 1/3 volume of killing buffer in it. The tubes were centrifuged at 5000 rpm for 5 minutes to obtain the pellets of bacteria which was immediately frozen using liquid nitrogen and subsequently stored in -80 o C until further use. Norfloxacin treatment of LZ strains was performed as described below. Norfloxacin at a final concentration of 20 μg/ml was added to mid exponentially growing cells at OD600 around 0.6 to induce the supercoiling changes. After addition of norfloxacin the cultures were incubated for another 5 minutes at 30 o C with shaking, before being sampled as described above. 2.2.11 Extraction and quality check of total RNA Total RNA from E.coli cells, used here mainly for synthesis of cDNA for various applications such as microarray hybridization, PCR and for primer extension experiments, was extracted using RNeasy Mini kit following the manufacturers instruction. To eliminate even small DNA contamination from RNA preparations, DNase treatment was performed twice. The first DNase treatment was done on the RNeasy column (part of the RNeasy kit protocol) and once after the final elution from the column, using DNA-free from Ambion. Handling of RNA was done using nuclease free tips and tubes from Ambion. All the buffers and water used were RNase free procured either as a part of the kit or from Ambion (Buffer kit, catalog number 9010). After the second DNase treatment, the RNA solution was extracted once by 27
MATERIALS AND METHODS phenol: chloroform method as described above to get rid of DNase removal reagent. The RNA was then quantified and precipitated by ethanol precipitation and then redissolved in appropriate volume of nuclease free water so as to obtain a RNA concentration appropriate for the application (for microarrays the concentration was maintained at around 6 μg/ml and for all other applications at 1 μg/ml). The quality of the RNA was checked by agarose gel electrophoresis and Agilent 2100 Bioanalyzer. 2.2.12 Microarray hybridization Whole genome E.coli K12 cDNA microarrays used in this study were purchased from MWG biotech (E.coli K12 arrays) & Ocimum biosolutions (E.coli K12 OciChip). The cDNA synthesis was performed as described in the MWG/Ocimum’s standardized protocols. After cDNA synthesis, the hybridization was performed in a moist chamber, at a temperature of 42 o C and with a mild shaking (40 rpm) for 16 to 18 hours. After hybridization the microarray slides are subjected to three consecutive washing steps as describe in the manual. Then the slides were dried and scanned at two wavelengths, 550nm for Cy3 and 640nm for Cy5, using ScanExpress confocal laser scanner (PerkinElmer). The scanner generates TIFF images of the microarray with individual spots showing relative amounts of Cy3 and Cy5 in each spot. Images were quantified using freely available Spotfinder software from TIGR [Saeed et al., 2003]. Figure 9 shows a schematic representation of the microarray experiment. The experiment was always carried out with two biological and technical replicates. The wild-type and the fis comparison shown below when repeated by interchanging the Cy3 to fis mutant and Cy5 to wild-type forms a technical replicate while a biological replicate is when the whole microarray experiment is repeated anew staring from the bacterial culture. Each biological replicate consists of at least one technical replicate. 28
Page 1 and 2: Coordinated regulation of gene expr
Page 3 and 4: Parts of this work has been publish
Page 5 and 6: ABSTRACT promoter region determines
Page 7 and 8: ACKNOWLEDGEMENTS first impression o
Page 9 and 10: TABLE OF CONTENTS 2.2.5 Preparation
Page 11 and 12: LIST OF TABLES List of tables Table
Page 13 and 14: LIST OF FIGURES List of Figures Fig
Page 15 and 16: ABBREVIATIONS Abbreviations ∆61
Page 17 and 18: ABBREVIATIONS TBE Tris TS tyrT35U t
Page 19 and 20: INTRODUCTION Salmonella: “In more
Page 21 and 22: INTRODUCTION Figure 1: Schematic il
Page 23 and 24: INTRODUCTION UP element -35 -10 Spa
Page 25 and 26: INTRODUCTION can also influence elo
Page 27 and 28: INTRODUCTION have more base-pairs t
Page 29 and 30: INTRODUCTION Figure 6: Graphical re
Page 31 and 32: INTRODUCTION shown that H-NS reache
Page 33 and 34: INTRODUCTION formed as a repercussi
Page 35 and 36: MATERIALS AND METHODS 2 Materials a
Page 37 and 38: MATERIALS AND METHODS NaCl 10 gm YT
Page 39 and 40: MATERIALS AND METHODS LZ41 3 pyrF28
Page 41 and 42: MATERIALS AND METHODS 2.2 Methods 2
Page 43: MATERIALS AND METHODS given using a
Page 47 and 48: MATERIALS AND METHODS Filtered (by
Page 49 and 50: MATERIALS AND METHODS free water (A
Page 51 and 52: RESULTS 3 Results Growth phase-depe
Page 53 and 54: RESULTS Neither the fis nor the hns
Page 55 and 56: RESULTS these identified genes (by
Page 57 and 58: RESULTS furthermore the colour of t
Page 59 and 60: RESULTS During the entire growth cy
Page 61 and 62: RESULTS transcripts of a growth pha
Page 63 and 64: RESULTS phosphorylation, are associ
Page 65 and 66: RESULTS Table 4: RT-PCR analysis of
Page 67 and 68: RESULTS Figure 16: Schematic repres
Page 69 and 70: RESULTS parC) which allows the supe
Page 71 and 72: RESULTS Figure 19: In vitro transcr
Page 73 and 74: RESULTS contrast the tyrTD promoter
Page 75 and 76: DISCUSSION 4 Discussion The idea of
Page 77 and 78: DISCUSSION In a study by Balke & Gr
Page 79 and 80: DISCUSSION cells maintain viability
Page 81 and 82: DISCUSSION writhe in the UAS by FIS
Page 83 and 84: DISCUSSION 4.3.2 Mechanism of trans
Page 85 and 86: CONCLUSION 5 Conclusion Regulation
Page 87 and 88: REFERENCES Bordes, P., Conter, A.,
Page 89 and 90: REFERENCES Gruber, T.M., and Gross,
Page 91 and 92: REFERENCES molecular basis for sele
Page 93 and 94: REFERENCES Pan, C.Q., Finkel, S.E.,
Page 95 and 96:
REFERENCES Schroder, O., and Wagner
Page 97 and 98:
REFERENCES Willenbrock, H., and Uss
Page 99 and 100:
APPENDIX I gene blattner ratio p-va
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
APPENDIX I Supplementary table - VI
Page 129 and 130:
APPENDIX I fabB b2323 0.6336 0.0043
Page 131 and 132:
APPENDIX I ibpA b3687 2.4960 0.0181
Page 133 and 134:
APPENDIX I pepP b2908 1.2182 0.0368
Page 135 and 136:
APPENDIX I speG b1584 0.4151 0.0001
Page 137 and 138:
APPENDIX I ycjX b1321 4.0841 0.0141
Page 139 and 140:
APPENDIX I yhfY b3382 1.4063 0.0453
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
APPENDIX I gene blattner ratio A/B
Page 159 and 160:
Page 161 and 162:
Page 163 and 164:
Page 165 and 166:
Page 167 and 168:
Page 169 and 170:
Page 171 and 172:
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
Page 179 and 180:
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
Page 191 and 192:
Page 193 and 194:
Page 195 and 196:
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
Statement of sources Declaration I
Page 207:
190
show all

Coordinated regulation of gene expression by E ... - Jacobs University

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?