by Cheung, Man Wai William. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 113-123). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.ii / DEDICATION --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- General Introduction --- p.1 / Chapter 1.2. --- Purpose of Study --- p.3 / Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Central Pathways of Aerobic and Anaerobic Carbon Catabolism --- p.7 / Chapter 2.2. --- Global Regulation of Gene Expression by Oxygen --- p.10 / Chapter 2.2.1. --- Two approaches for the studies --- p.10 / Chapter 2.2.2. --- FNR regulation --- p.12 / Chapter 2.2.3. --- ArcAB regulation --- p.19 / Chapter 2.2.3.1. --- arcA --- p.19 / Chapter 2.2.3.2. --- arcB --- p.20 / Chapter 2.2.3.3. --- A member of the Two- Components regulatory systems --- p.21 / Chapter 2.3. --- Molecular Analysis of Promoters --- p.26 / Chapter 2.3.1. --- S1 mapping --- p.29 / Chapter 2.3.2. --- Primer extension --- p.29 / Chapter 2.3.3. --- DNaseI footprinting --- p.30 / Chapter 2.3.4. --- Mutational analysis of promoters --- p.32 / Chapter 3. --- Materials and Methods --- p.35 / Chapter 3.1. --- Bacterial strains and Plasmids --- p.35 / Chapter 3.2. --- Media --- p.35 / Chapter 3.3. --- Solutions --- p.38 / Chapter 3.4. --- Small Scale Preparation of Plasmid DNA --- p.40 / Chapter 3.5. --- Large Scale Preparation of Plasmid DNA --- p.41 / Chapter 3.5.1. --- Growth of bacterial culture --- p.41 / Chapter 3.5.2. --- Lysis by alkali --- p.43 / Chapter 3.5.3. --- Purification of closed circular DNA by cesium chloride gradient equilibrium centrifugation --- p.44 / Chapter 3.5.4. --- Digestion of DNA with restriction endonucleases --- p.45 / Chapter 3.6. --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.45 / Chapter 3.7. --- Cloning of DNA Fragments from Nest-deleted M13mpl8 Clones to pFZYl --- p.47 / Chapter 3.8. --- Introduction of Plasmids into Cells --- p.48 / Chapter 3.8.1. --- Heat shock transformation --- p.48 / Chapter 3.8.1.1. --- Preparation of competent cells (I) --- p.48 / Chapter 3.8.1.2. --- Preparation of competent cells (II) --- p.49 / Chapter 3.8.2. --- High efficiency transformation by electroporation --- p.50 / Chapter 3.8.2.1. --- Preparation of electro- competent cells --- p.50 / Chapter 3.8.2.2. --- Electro-transformation --- p.51 / Chapter 3.9. --- DNA Sequencing by Chain Termination Method --- p.51 / Chapter 3.9.1. --- Preparation of single-stranded M13 templates for sequencing reaction --- p.51 / Chapter 3.9.2. --- Sequencing reactions using single- stranded templates --- p.53 / Chapter 3.9.3. --- Preparation of polyacrylamide gel for sequencing --- p.54 / Chapter 3.9.4. --- Electrophoresis of the DNA samples --- p.55 / Chapter 3.10. --- Construction of Nested Clones by Exonuclease III Unidirectional Deletions --- p.55 / Chapter 3.10.1. --- Unidirectional nested deletion of M13mpl8 clones --- p.55 / Chapter 3.10.2. --- Screening of nested clones by Direct gel electrophoresis --- p.56 / Chapter 3.10.3. --- Screening of nested clones of M13mpl8 and pFZYl by Polymerase Chain Reaction --- p.57 / Chapter 3.11. --- β-galactosidase Assay --- p.59 / Chapter 3.12. --- Primer Extension --- p.60 / Chapter 3.12.1. --- Preparation of total RNA from Gram- negative bacteria --- p.60 / Chapter 3.12.2. --- Labelling the 5' end of the oligonucleotides --- p.61 / Chapter 3.12.3. --- Hybridization and primer extension --- p.62 / Chapter 4. --- Result --- p.63 / Chapter 4.1. --- Subcloning of arcA promoter into M13mpl8/19 --- p.63 / Chapter 4.2. --- Sequencing of p34一18i and p3419i using M13 Sequencing primers (-47) and ArcA-cds Primers --- p.63 / Chapter 4.3. --- Unidirectional Nested Deletion of p3418i using Exonuclease III --- p.65 / Chapter 4.3.1. --- Large scale preparation of p3A18i DNA for Exonuclease III unidirectional nested deletion --- p.65 / Chapter 4.3.2. --- Construction of 3' and 5' overhangs --- p.65 / Chapter 4.3.3. --- Exonuclease III digestion --- p.67 / Chapter 4.3.4. --- Repairing of the 3' and 5' overhangs to generate blunt ends --- p.67 / Chapter 4.3.5. --- Blunt-end ligation of the nested deletion M13mpl8 subclones p3418i --- p.67 / Chapter 4.3.6. --- Transformation --- p.69 / Chapter 4.3.7. --- Screening of nest-deleted p3418i clones by Direct Gel --- p.71 / Chapter 4.3.8. --- Screening of nested deletion p3418i clones by PCR Screening --- p.73 / Chapter 4.3.9. --- Sequencing of the nested deletion p3418i clones --- p.76 / Chapter 4.4. --- Cloning of Nested Deletion DNA Fragments from M13mpl8 into pFZYl --- p.80 / Chapter 4.4.1. --- Screening of pFZYl clones using PCR Screening --- p.80 / Chapter 4.5. --- Expression of Nest-Deleted arcA Promoter Clones in E. coli MC1061-5 --- p.87 / Chapter 4.6. --- Expression of Nest-Deleted arcA Promoter Clones in S. typhimurium JR501 --- p.89 / Chapter 4.7. --- Primer Extension --- p.89 / Chapter 5. --- Discussion --- p.93 / Chapter 5.1. --- Sequencing of arcA Promoter --- p.93 / Chapter 5.2. --- Unidirectional Nested Deletion of p3A18i using Exonuclease III --- p.94 / Chapter 5.3. --- Screening of Nest-deletion p3418i Subclones --- p.95 / Chapter 5.4 --- Cloning of Nest-deleted DNA Fragments from M13mpl8 Subclones into pFZYl --- p.99 / Chapter 5.5. --- Screening of Nest-deleted pFZYl Subclones of p3418i --- p.101 / Chapter 5.6. --- The Effect of 5' Unidirectional Nested Deletion on the Expression of the Cloned arcA promoter in E. coli M1061-5 and S typhimurium JR501 --- p.102 / Chapter 5.7. --- Primer Extension --- p.102 / Chapter 5.8. --- Sequence Analysis of the Cloned arcA Promoter --- p.104 / Chapter 6. --- Conclusion and Further Studies --- p.111 / Chapter 7. --- Reference Cited --- p.113
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318929 |
| Date | January 1992 |
| Contributors | Cheung, Man Wai., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, x, 123 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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