Geok-yen Yeo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 225-233). / Title page --- p.i / Members of Thesis Advisory Committee --- p.ii / Abstract --- p.iii -iv / Acknowledgments --- p.v / Dedication --- p.vi / Table of Contents --- p.vii -xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-31 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Fermentation --- p.1 / Chapter 1.3 --- Growth in Escherichia coli --- p.3 / Chapter 1.3.1 --- Aerobic growth in Escherichia coli --- p.3 / Chapter 1.3.2 --- The regulation of enzyme synthesis during cell metabolism --- p.7 / Chapter 1.3.3 --- Anaerobic growth in E. coli --- p.8 / Chapter 1.3.4 --- Anaerobic regulation by the transcriptional regulator Fnr --- p.12 / Chapter 1.3.5 --- "The case for ""Pasteur Control Proteins"" (PCP)" --- p.13 / Chapter 1.4 --- The family of alcohol dehydrogenases : An overview --- p.15 / Chapter 1.4.1 --- Molecular characteristics of alcohol dehydrogenases --- p.17 / Chapter 1.4.2 --- Residue conservation in alcohol dehydrogenases --- p.24 / Chapter 1.4.3 --- The effect of amino acid substitution on substrate specificity --- p.25 / Chapter 1.5 --- Alcohol dehydrogenases in bacteria --- p.28 / Chapter 1.5.1 --- Alcohol dehydrogenase in E. coli --- p.28 / Chapter 1.6 --- Aims of this study --- p.30 / Chapter CHAPTER 2 --- MATERIALS & METHODS --- p.32 -90 / Chapter 2.1 --- Bacterial strains --- p.32 / Chapter 2.2 --- Plasmids --- p.32 / Chapter 2.2.1 --- "Low copy number plasmid, pTJS75Km" --- p.32 / Chapter 2.2.2 --- "High copy number plasmid, pUC18" --- p.33 / Chapter 2.3 --- Bacterial culture media and solutions --- p.39 / Chapter 2.3.1 --- Luria Bertani (LB) medium --- p.39 / Chapter 2.3.2 --- L-Broth + MOPS --- p.39 / Chapter 2.3.3 --- "R medium, containing Triphenyltetrazolium chloride-ethanol (TTC-EtOH)" --- p.40 / Chapter 2.3.4 --- SOB and SOC media --- p.41 / Chapter 2.3.5 --- M9 Glucose medium --- p.42 / Chapter 2.3.6 --- Terrific Broth (TB) --- p.42 / Chapter 2.3.7 --- Rich Broth (RB) --- p.43 / Chapter 2.3.8 --- Antibiotic solutions --- p.43 / Chapter 2.4 --- Restriction endonucleases and other enzymes --- p.44 / Chapter 2.5 --- Isolation of chromosomal DNA --- p.45 / Chapter 2.5.1 --- Preparation of chromosomal DNA by spooling --- p.45 / Chapter 2.5.2 --- Preparation of chromosomal DNA by cesium chloride density gradient --- p.48 / Chapter 2.6 --- Isolation of plasmid DNA --- p.50 / Chapter 2.6.1 --- Large-scale preparation of plasmid by CsCl density gradient --- p.50 / Chapter 2.6.2 --- Small-scale preparation of plasmid DNA --- p.54 / Chapter 2.6.2. --- A Boiling method --- p.54 / Chapter 2.6.2. --- B Alkaline Lysis method --- p.55 / Chapter 2.6.3 --- Preparation of plasmid DNA by Qiagen columns --- p.56 / Chapter 2.7 --- Purification of DNA --- p.59 / Chapter 2.7.1 --- Ethanol precipitation --- p.59 / Chapter 2.7.2 --- Concentration and desalting using Centricon columns --- p.59 / Chapter 2.7.3 --- Purification of DNA by Geneclean procedure --- p.61 / Chapter 2.8 --- DNA cloning techniques --- p.63 / Chapter 2.8.1 --- Restriction endonuclease digestion --- p.63 / Chapter 2.8.2 --- Agarose-ethidium bromide gel electrophoresis --- p.65 / Chapter 2.8.2. --- A Gel loading buffer --- p.66 / Chapter 2.8.2. --- B Electro-elution of DNA --- p.67 / Chapter 2.8.3 --- Size fractionation --- p.68 / Chapter 2.8.3. --- A Salt gradient fractionation --- p.68 / Chapter 2.8.3. --- B Sucrose gradient --- p.70 / Chapter 2.8.4 --- Dephosphorylation of restriction-enzyme digested vector plasmid using calf intestinal phosphatase (CIP) --- p.71 / Chapter 2.8.5 --- Ligation of vector and insert --- p.72 / Chapter 2.8.6 --- Preparation of competent cells --- p.73 / Chapter 2.8.7 --- DNA transformation --- p.75 / Chapter 2.8.7.A --- By heat shock --- p.75 / Chapter 2.8.7.B --- By electroporation --- p.75 / Chapter 2.9 --- Screening for adhC transformants --- p.78 / Chapter 2.9.1 --- Screening for adhC clones --- p.78 / Chapter 2.9.2 --- Screening for pUC18 transformants --- p.79 / Chapter 2.10 --- Confirmation of adhC clones --- p.80 / Chapter 2.10.1 --- Reproduction of red colonies on R plates and antibiotic resistance --- p.80 / Chapter 2.10.2 --- T7 phage test for E. coli strains --- p.80 / Chapter 2.10.3 --- Plasmid size determination --- p.82 / Chapter 2.10.4 --- Re-transformation into E. coli host strains --- p.82 / Chapter 2.10.5 --- Physiological study of adhC clones --- p.83 / Chapter 2.10.6 --- Alcohol dehydrogenase assay --- p.84 / Chapter 2.11 --- The dye-binding method of protein determination --- p.87 / Chapter 2.12 --- Special procedures --- p.88 / Chapter 2.12.1 --- Generation of adh clones with deletions --- p.88 / Chapter 2.12.2 --- Sequencing reactions --- p.89 / Chapter CHAPTER 3 --- RESULTS: PART I Cloning and Restriction Mapping of the adhC mutation in a low copy number plasmid vector --- p.91 -122 / Chapter 3.1 --- Introduction: Cloning strategy --- p.91 / Chapter 3.2 --- Cloning of the adh mutation from strain CC2807B (an ADH overproducing mutant strain) in pTJS75Km --- p.93 / Chapter 3.2.1 --- Construction of the 'HK' clones --- p.93 / Chapter 3.3 --- Restriction mapping of the adh clones --- p.101 / Chapter 3.4 --- Subcloning the adhC insert --- p.110 / Chapter 3.4.1 --- Construction of plasmid pHK14 --- p.110 / Chapter 3.4.2 --- Construction of plasmid pHK15 --- p.115 / Chapter 3.4.3 --- Construction of plasmid pSS22 --- p.121 / Chapter 3.5 --- Remarks concerning the clones --- p.121 / Chapter CHAPTER 4 --- RESULTS:PART II Cloning and Sequencing of the adhC mutation in a high copy number plasmid vector --- p.123 -148 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.1.1 --- Choice of sequencing strategy --- p.123 / Chapter 4.1.2 --- An attempt to eliminate clone instability --- p.124 / Chapter 4.2 --- Subcloning of adh insert in pUC18 --- p.125 / Chapter 4.2.1 --- Study of adh clone EPR --- p.125 / Chapter 4.2.2 --- Re-construction of plasmid pEPR ( = pEE5) --- p.126 / Chapter 4.2.3 --- Construction of plasmids pEH2 and pEH3 --- p.127 / Chapter 4.2.4 --- Construction of a nested deletion library --- p.138 / Chapter CHAPTER 5 --- RESULTS : PART III Sequencing of the Mutation --- p.149 -177 / Chapter 5.1 --- Nucleotide sequencing --- p.149 / Chapter 5.2 --- Sequencing of the cloned adhC gene insert --- p.150 / Chapter 5.3 --- Analysis of the sequenced DNA by DNASIS computer software --- p.151 / Chapter 5.3.1 --- Search for codons associated with initiation and termination of transcription using the open reading frame (ORF) search --- p.151 / Chapter 5.3.2 --- Translation of the nucleotide sequence at the open reading frame (start 223 - end 2896) --- p.152 / Chapter 5.4 --- Search for DNA sequence homology with known DNA sequences --- p.152 / Chapter 5.4.1 --- Sequence homology of the structural gene (nucleotide # 223- #28%) : Two nucleotide changes revealed in DNA sequence of the structural gene adhE of Escherichia coli --- p.153 / Chapter 5.4.2 --- adhC mutation is due to changes in two amino acids --- p.153 / Chapter 5.4.3 --- The DNA sequence 5' of the mutated structural gene (upstream sequence) --- p.155 / Chapter 5.4.4 --- The DNA sequence 3' of the mutated structural gene (downstream sequence) --- p.156 / Chapter 5.5 --- Comparisons between the computer-predicted properties of the mutant and wild-type protein --- p.156 / Chapter 5.5.1 --- Prediction of the alcohol dehydrogenase protein secondary structure by the Robson Method --- p.156 / Chapter 5.5.2 --- Isoelectric point prediction --- p.156 / Chapter CHAPTER 6 --- RESULTS : PART IV Comparative Studies of Alcohol Dehydrogenase Expressionin adhC Strains and Clones --- p.178 -203 / Chapter 6.1 --- Introduction --- p.178 / Chapter 6.1.1 --- Basis for the alcohol dehydrogenase assay --- p.178 / Chapter 6.1.2 --- Choice of assay method --- p.179 / Chapter 6.1.3 --- Points to consider for ADH assay --- p.179 / Chapter 6.2 --- General growth characteristics of bacterial strains --- p.181 / Chapter 6.2.1 --- Plate cultures --- p.181 / Chapter 6.2.2 --- Overnight liquid cultures --- p.183 / Chapter 6.2.3 --- Batch liquid cultures --- p.183 / Chapter 6.2.4 --- ADH activity of strain CC2807B --- p.190 / Chapter 6.2.5 --- Comparison of ADH activity --- p.192 / Chapter 6.3 --- Investigating the mutated ADH enzyme --- p.197 / Chapter 6.3.1 --- Oxygen inactivation of the mutated enzyme --- p.197 / Chapter 6.3.2 --- Thermostability of the mutated enzyme --- p.201 / Chapter CHAPTER 7 --- DISCUSSION --- p.204 -220 / Chapter 7.1 --- Cloning of the adhC mutation --- p.204 / Chapter 7.1.1 --- Instability of clones in plasmid vector pUC18 --- p.204 / Chapter 7.1.2 --- Eliminating 'toxic' genes adjacent to adh locus --- p.207 / Chapter 7.1.3 --- Cloning in pTJS75Km low copy number vector --- p.208 / Chapter 7.2 --- DNA sequence of the adhC clones --- p.211 / Chapter 7.2.1 --- The basis for sequencing pUC 18-derived clones --- p.211 / Chapter 7.2.2 --- Homology to known alcohol dehydrogenases (ADH) sequences --- p.213 / Chapter 7.3 --- Findings concerning the adhC mutation --- p.217 / Chapter 7.3.1 --- How amino acid substitutions may affect an enzyme --- p.217 / Chapter 7.3.2 --- Physiological aspects of the bacterial cell due to the mutated enzyme --- p.218 / Chapter 7.4 --- Conclusions --- p.220 / APPENDICES --- p.221 -224 / REFERENCES --- p.225 -233
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318078 |
| Date | January 1994 |
| Contributors | Yeo, Geok-yen., 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, xi, 233 leaves : ill. (some mounted col.) ; 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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