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

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MATERIALS AND METHODS ptyrTS [Schneider et al., 2000b] ∆61 constructs of ptyrTlac, ptyrTD, ptyrT35U, ptyrTS [Auner et al., 2003] pUC18 [Yanisch-Perron et al., 1985] 2.1.7 Primers Primers for RT-PCR: Forward Reverse aceB CGAGCAGGAGAAGCAAATTC GCGCTGCCAGAAGTTTATTG glcB TATACGGCAGCGACATCATC CAGCGGTAGAGATTCATCGAG purC TGATCGCAAAGGTATGGTGA AGCAGACGCTCCATTTGAGT purF ATGCCTTAACGGTGCTTCAG GGCGAGCTTCAAATACATCG purK AAAGCGTGATTACCGCTGAG sdhD CGTTCACCAAAGTGTTCACC GGTCAGCAATAATCGGGAAC CCAGCGGTTTAACGTAGTCG sucC CAAAGTGAAAGCCGTTCTGG AACACCCACTTCTGCTACCG Primers for primer extension: Ex103 GGAATGTGTAAGAGCGGGG G11 bla GAGCAGTGCCGCTTCGC CAGGAAGGCAAAATGCCCC 23
MATERIALS AND METHODS 2.2 Methods 2.2.1 Agarose gel electrophoresis Electrophoretic separation of DNA was carried out using 0.8-1.5% (w/v) agarose gels prepared in 1X Tris-borate (TBE) or Tris-acetate buffer (TAE). The gel buffer and running buffer are identical. For all applications (except for topology gels) ethidium bromide (0.3 μg/ml) was mixed with the gel before casting. Various size of gel chambers (8 X 10 to 19.5 X 19.5 cm) were used to cast the gel and the DNA to be separated was mixed with 1/6 th the volume of loading dye, with size markers (λ pst, see below) when necessary. Separation was carried out at 0.5 V/cm for topology gels and at 1-5 V/cm for other purposes. Subsequently gels were observed under UV light in gel documentation equipment (Biodoc analyze from Biometra ® ) and photographed when necessary. DNA ladder for size marker was obtained by digesting overnight 25 μg of λ DNA (New England Biolabs) with PstI restriction enzyme in an appropriate buffer at 37 o C. 2.2.2 Small and medium scale isolation of plasmid DNA using Qiagen kit Small and medium scale plasmid isolation was performed using QIAprep Spin Miniprep Kit and QIAGEN Plasmid Maxi Kit respectively by following the manufacturer’s instruction. The E.coli cultured in 2X YT and LB has been used for extracting plasmid. 24
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: MATERIALS AND METHODS LZ41 3 pyrF28
Page 43 and 44: MATERIALS AND METHODS given using a
Page 45 and 46: MATERIALS AND METHODS phenol: chlor
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
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REFERENCES Pan, C.Q., Finkel, S.E.,
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REFERENCES Schroder, O., and Wagner
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REFERENCES Willenbrock, H., and Uss
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APPENDIX I gene blattner ratio p-va
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APPENDIX I Supplementary table - VI
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APPENDIX I fabB b2323 0.6336 0.0043
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APPENDIX I ibpA b3687 2.4960 0.0181
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APPENDIX I pepP b2908 1.2182 0.0368
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APPENDIX I speG b1584 0.4151 0.0001
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APPENDIX I ycjX b1321 4.0841 0.0141
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APPENDIX I yhfY b3382 1.4063 0.0453
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APPENDIX I gene blattner ratio A/B
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Statement of sources Declaration I
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