Studies of the recombinant plasmids carrying the adh mutation of escherichia coli.

Geok-yen Yeo.Thesis (M.Phil.)--Chinese University of Hong Kong, 1994.Includes bibliographical references (leaves 225-233).Title page --- p.iMembers of Thesis Advisory Committee --- p.iiAbstract --- p.iii -ivAcknowledgments --- p.vDedication --- p.viTable of Contents --- p.vii -xiChapter CHAPTER 1 --- INTRODUCTION --- p.1-31Chapter 1.1 --- General Introduction --- p.1Chapter 1.2 --- Fermentation --- p.1Chapter 1.3 --- Growth in Escherichia coli --- p.3Chapter 1.3.1 --- Aerobic growth in Escherichia coli --- p.3Chapter 1.3.2 --- The regulation of enzyme synthesis during cell metabolism --- p.7Chapter 1.3.3 --- Anaerobic growth in E. coli --- p.8Chapter 1.3.4 --- Anaerobic regulation by the transcriptional regulator Fnr --- p.12Chapter 1.3.5 --- "The case for ""Pasteur Control Proteins"" (PCP)" --- p.13Chapter 1.4 --- The family of alcohol dehydrogenases : An overview --- p.15Chapter 1.4.1 --- Molecular characteristics of alcohol dehydrogenases --- p.17Chapter 1.4.2 --- Residue conservation in alcohol dehydrogenases --- p.24Chapter 1.4.3 --- The effect of amino acid substitution on substrate specificity --- p.25Chapter 1.5 --- Alcohol dehydrogenases in bacteria --- p.28Chapter 1.5.1 --- Alcohol dehydrogenase in E. coli --- p.28Chapter 1.6 --- Aims of this study --- p.30Chapter CHAPTER 2 --- MATERIALS & METHODS --- p.32 -90Chapter 2.1 --- Bacterial strains --- p.32Chapter 2.2 --- Plasmids --- p.32Chapter 2.2.1 --- "Low copy number plasmid, pTJS75Km" --- p.32Chapter 2.2.2 --- "High copy number plasmid, pUC18" --- p.33Chapter 2.3 --- Bacterial culture media and solutions --- p.39Chapter 2.3.1 --- Luria Bertani (LB) medium --- p.39Chapter 2.3.2 --- L-Broth + MOPS --- p.39Chapter 2.3.3 --- "R medium, containing Triphenyltetrazolium chloride-ethanol (TTC-EtOH)" --- p.40Chapter 2.3.4 --- SOB and SOC media --- p.41Chapter 2.3.5 --- M9 Glucose medium --- p.42Chapter 2.3.6 --- Terrific Broth (TB) --- p.42Chapter 2.3.7 --- Rich Broth (RB) --- p.43Chapter 2.3.8 --- Antibiotic solutions --- p.43Chapter 2.4 --- Restriction endonucleases and other enzymes --- p.44Chapter 2.5 --- Isolation of chromosomal DNA --- p.45Chapter 2.5.1 --- Preparation of chromosomal DNA by spooling --- p.45Chapter 2.5.2 --- Preparation of chromosomal DNA by cesium chloride density gradient --- p.48Chapter 2.6 --- Isolation of plasmid DNA --- p.50Chapter 2.6.1 --- Large-scale preparation of plasmid by CsCl density gradient --- p.50Chapter 2.6.2 --- Small-scale preparation of plasmid DNA --- p.54Chapter 2.6.2. --- A Boiling method --- p.54Chapter 2.6.2. --- B Alkaline Lysis method --- p.55Chapter 2.6.3 --- Preparation of plasmid DNA by Qiagen columns --- p.56Chapter 2.7 --- Purification of DNA --- p.59Chapter 2.7.1 --- Ethanol precipitation --- p.59Chapter 2.7.2 --- Concentration and desalting using Centricon columns --- p.59Chapter 2.7.3 --- Purification of DNA by Geneclean procedure --- p.61Chapter 2.8 --- DNA cloning techniques --- p.63Chapter 2.8.1 --- Restriction endonuclease digestion --- p.63Chapter 2.8.2 --- Agarose-ethidium bromide gel electrophoresis --- p.65Chapter 2.8.2. --- A Gel loading buffer --- p.66Chapter 2.8.2. --- B Electro-elution of DNA --- p.67Chapter 2.8.3 --- Size fractionation --- p.68Chapter 2.8.3. --- A Salt gradient fractionation --- p.68Chapter 2.8.3. --- B Sucrose gradient --- p.70Chapter 2.8.4 --- Dephosphorylation of restriction-enzyme digested vector plasmid using calf intestinal phosphatase (CIP) --- p.71Chapter 2.8.5 --- Ligation of vector and insert --- p.72Chapter 2.8.6 --- Preparation of competent cells --- p.73Chapter 2.8.7 --- DNA transformation --- p.75Chapter 2.8.7.A --- By heat shock --- p.75Chapter 2.8.7.B --- By electroporation --- p.75Chapter 2.9 --- Screening for adhC transformants --- p.78Chapter 2.9.1 --- Screening for adhC clones --- p.78Chapter 2.9.2 --- Screening for pUC18 transformants --- p.79Chapter 2.10 --- Confirmation of adhC clones --- p.80Chapter 2.10.1 --- Reproduction of red colonies on R plates and antibiotic resistance --- p.80Chapter 2.10.2 --- T7 phage test for E. coli strains --- p.80Chapter 2.10.3 --- Plasmid size determination --- p.82Chapter 2.10.4 --- Re-transformation into E. coli host strains --- p.82Chapter 2.10.5 --- Physiological study of adhC clones --- p.83Chapter 2.10.6 --- Alcohol dehydrogenase assay --- p.84Chapter 2.11 --- The dye-binding method of protein determination --- p.87Chapter 2.12 --- Special procedures --- p.88Chapter 2.12.1 --- Generation of adh clones with deletions --- p.88Chapter 2.12.2 --- Sequencing reactions --- p.89Chapter CHAPTER 3 --- RESULTS: PART I Cloning and Restriction Mapping of the adhC mutation in a low copy number plasmid vector --- p.91 -122Chapter 3.1 --- Introduction: Cloning strategy --- p.91Chapter 3.2 --- Cloning of the adh mutation from strain CC2807B (an ADH overproducing mutant strain) in pTJS75Km --- p.93Chapter 3.2.1 --- Construction of the 'HK' clones --- p.93Chapter 3.3 --- Restriction mapping of the adh clones --- p.101Chapter 3.4 --- Subcloning the adhC insert --- p.110Chapter 3.4.1 --- Construction of plasmid pHK14 --- p.110Chapter 3.4.2 --- Construction of plasmid pHK15 --- p.115Chapter 3.4.3 --- Construction of plasmid pSS22 --- p.121Chapter 3.5 --- Remarks concerning the clones --- p.121Chapter CHAPTER 4 --- RESULTS:PART II Cloning and Sequencing of the adhC mutation in a high copy number plasmid vector --- p.123 -148Chapter 4.1 --- Introduction --- p.123Chapter 4.1.1 --- Choice of sequencing strategy --- p.123Chapter 4.1.2 --- An attempt to eliminate clone instability --- p.124Chapter 4.2 --- Subcloning of adh insert in pUC18 --- p.125Chapter 4.2.1 --- Study of adh clone EPR --- p.125Chapter 4.2.2 --- Re-construction of plasmid pEPR ( = pEE5) --- p.126Chapter 4.2.3 --- Construction of plasmids pEH2 and pEH3 --- p.127Chapter 4.2.4 --- Construction of a nested deletion library --- p.138Chapter CHAPTER 5 --- RESULTS : PART III Sequencing of the Mutation --- p.149 -177Chapter 5.1 --- Nucleotide sequencing --- p.149Chapter 5.2 --- Sequencing of the cloned adhC gene insert --- p.150Chapter 5.3 --- Analysis of the sequenced DNA by DNASIS computer software --- p.151Chapter 5.3.1 --- Search for codons associated with initiation and termination of transcription using the open reading frame (ORF) search --- p.151Chapter 5.3.2 --- Translation of the nucleotide sequence at the open reading frame (start 223 - end 2896) --- p.152Chapter 5.4 --- Search for DNA sequence homology with known DNA sequences --- p.152Chapter 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.153Chapter 5.4.2 --- adhC mutation is due to changes in two amino acids --- p.153Chapter 5.4.3 --- The DNA sequence 5' of the mutated structural gene (upstream sequence) --- p.155Chapter 5.4.4 --- The DNA sequence 3' of the mutated structural gene (downstream sequence) --- p.156Chapter 5.5 --- Comparisons between the computer-predicted properties of the mutant and wild-type protein --- p.156Chapter 5.5.1 --- Prediction of the alcohol dehydrogenase protein secondary structure by the Robson Method --- p.156Chapter 5.5.2 --- Isoelectric point prediction --- p.156Chapter CHAPTER 6 --- RESULTS : PART IV Comparative Studies of Alcohol Dehydrogenase Expressionin adhC Strains and Clones --- p.178 -203Chapter 6.1 --- Introduction --- p.178Chapter 6.1.1 --- Basis for the alcohol dehydrogenase assay --- p.178Chapter 6.1.2 --- Choice of assay method --- p.179Chapter 6.1.3 --- Points to consider for ADH assay --- p.179Chapter 6.2 --- General growth characteristics of bacterial strains --- p.181Chapter 6.2.1 --- Plate cultures --- p.181Chapter 6.2.2 --- Overnight liquid cultures --- p.183Chapter 6.2.3 --- Batch liquid cultures --- p.183Chapter 6.2.4 --- ADH activity of strain CC2807B --- p.190Chapter 6.2.5 --- Comparison of ADH activity --- p.192Chapter 6.3 --- Investigating the mutated ADH enzyme --- p.197Chapter 6.3.1 --- Oxygen inactivation of the mutated enzyme --- p.197Chapter 6.3.2 --- Thermostability of the mutated enzyme --- p.201Chapter CHAPTER 7 --- DISCUSSION --- p.204 -220Chapter 7.1 --- Cloning of the adhC mutation --- p.204Chapter 7.1.1 --- Instability of clones in plasmid vector pUC18 --- p.204Chapter 7.1.2 --- Eliminating 'toxic' genes adjacent to adh locus --- p.207Chapter 7.1.3 --- Cloning in pTJS75Km low copy number vector --- p.208Chapter 7.2 --- DNA sequence of the adhC clones --- p.211Chapter 7.2.1 --- The basis for sequencing pUC 18-derived clones --- p.211Chapter 7.2.2 --- Homology to known alcohol dehydrogenases (ADH) sequences --- p.213Chapter 7.3 --- Findings concerning the adhC mutation --- p.217Chapter 7.3.1 --- How amino acid substitutions may affect an enzyme --- p.217Chapter 7.3.2 --- Physiological aspects of the bacterial cell due to the mutated enzyme --- p.218Chapter 7.4 --- Conclusions --- p.220APPENDICES --- p.221 -224REFERENCES --- p.225 -23