Contents 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA... 1 Introduction... 1 Principle... 1 Reagents Required and Their Role... 2 Procedure... 3 Observation... 4 Result Table... 4 Troubleshootings... 4 Precautions... 4 Exp. 1.2 Preparation of Bacterial Lysates... 5 Introduction... 5 Principle... 6 Procedure... 7 Observation... 9 Result Table... 9 Troubleshootings... 9 Precautions... 9 Exp. 1.3 Isolation of Plasmids... 12 Introduction... 12 Principle... 13 Reagents Required and Their Role... 13 Procedure... 15 Observation... 15 Result Table... 16 Troubleshootings... 16 Precautions... 16 Exp. 1.4 Isolation of Total RNA from Bacteria... 17 Introduction... 17 Principle... 18 Reagents Required and Their Role... 19 Procedure... 20 Observation... 20 Result Table... 21 Troubleshootings... 21 Precautions... 21 Exp. 1.5 Amplification of 16S rrna Gene... 22 vii
viii Contents Introduction... 22 Principle... 23 Reagents Required and Their Role... 25 Procedure... 26 Observation... 27 Troubleshootings... 28 Precautions... 28 Exp. 1.6 To Perform Agarose Gel Electrophoresis... 29 Introduction... 29 Principle... 30 Reagents Required and Their Role... 31 Procedure... 32 Observation... 33 Troubleshootings... 33 Precautions... 34 2 Cloning and Transformation... 35 Exp. 2.1 Preparation of Competent Cells and Heat-Shock Transformation... 35 Introduction... 35 Principle... 35 Reagents Required and Their Role... 37 Procedure... 38 Observation... 39 Troubleshooting... 39 Precautions... 39 Exp. 2.2 Electroporation... 41 Introduction... 41 Principle... 42 Reagents Required and Their Role... 43 Procedure... 43 Observation... 44 Result Table... 45 Troubleshooting... 45 Precautions... 45 Exp. 2.3 Restriction Digestion and Ligation... 46 Introduction... 46 Principle... 47 Reagents Required and Their Role... 50 Procedure... 51 Observation... 52 Troubleshooting... 52 Precaution... 53 Exp. 2.4 Selection of a Suitable Vector System for Cloning... 54 Different Types of Cloning Vectors... 55 Criteria for Choosing a Suitable Cloning Vector... 60 Conclusion... 62 Exp. 2.5 Confirmation of Transformation by Blue-White Selection... 62
Contents ix Introduction... 62 Principle... 63 Reagents Required and Their Role... 64 IPTG... 64 Antibiotics... 65 pbluescript... 65 Transformation Reaction Product... 65 Procedure... 65 Observation... 65 Troubleshooting... 66 Precautions... 66 Exp. 2.6 Confirmation of Cloning by PCR... 67 Introduction... 67 Principle... 68 Reagents Required and Their Role... 68 Procedure... 70 Observation... 70 Troubleshooting... 71 Precautions... 71 3 Advanced Molecular Microbiology Techniques... 73 Exp. 3.1. Synthesis of cdna... 73 Introduction... 73 Principle... 73 Reagents Required and Their Role... 75 Procedure... 76 Observation... 77 Trouble-Shootings... 78 Precautions... 78 Exp. 3.2. Gene Expression Analysis by qrt-pcr... 79 Introduction... 79 Principle... 80 Reagents Required and Their Role... 82 Procedure... 83 Observation... 84 Trouble-Shootings... 85 Precautions... 85 Exp. 3.3. Gene Expression Analysis Using Reporter Gene Assay... 86 Introduction... 86 Principle... 87 Reagents Required and Their Role... 87 Procedure... 88 Observation... 89 Result Table... 89 Precaution... 89 Trouble-Shootings... 89
x Contents Exp. 3.4. Semi-quantitative Gene Expression Analysis... 90 Introduction... 90 Principle... 91 Reagents Required and Their Role... 92 Procedure... 94 Observation... 94 Observation Table... 95 Trouble-Shootings... 96 Precautions... 96 Exp. 3.5. Northern Blotting... 97 Introduction... 97 Principle... 98 Reagents Required and Their Role... 99 Procedure... 100 Observation... 102 Trouble-Shootings... 102 Precautions... 103 Exp. 3.6. Isolation of Metagenomic DNA... 104 Introduction... 104 Principle... 105 Reagents Required and Their Role... 106 Procedure... 107 Observation... 108 Result Table... 108 Trouble-Shootings... 108 Precautions... 109 Exp. 3.7. Plasmid Curing from Bacterial Cell... 109 Introduction... 109 Principle... 110 Reagents Required and Their Role... 111 Procedure... 112 Observation... 112 Result Table... 112 Trouble-Shootings... 113 Precautions... 113 Exp. 3.8. Conjugation in Bacteria... 114 Introduction... 114 Principle... 114 Reagents Required and Their Role... 115 Procedure... 116 Observation... 116 Result Table... 117 Trouble-Shootings... 117 Precaution... 117 Exp. 3.9. Transduction in Bacteria... 118 Introduction... 118 Principle... 119 Reagents Required and Their Role... 120
Contents xi Procedure... 121 Observation... 122 Result Table... 122 Trouble-Shootings... 122 Precaution... 122 4 Molecular Microbial Diversity... 125 Exp. 4.1 Plasmid Profile Analysis... 125 Introduction... 125 Principle... 125 Reagents Required and Their Role... 126 Procedure... 128 Observation... 129 Result Table... 129 Troubleshooting... 132 Precautions... 132 Exp. 4.2 Amplified Ribosomal DNA Restriction Analysis to Study Bacterial Relatedness... 134 Introduction... 134 Principle... 135 Reagents Required and Their Role... 136 Procedure... 138 Observation... 139 Result Table... 142 Troubleshooting... 142 Precautions... 143 Exp. 4.3 Denaturing Gradient Gel Electrophoresis (DGGE) Analysis to Study Metagenomic Bacterial Diversity... 144 Introduction... 144 Principle... 145 Reagents Required and Their Role... 146 Procedure... 147 Observation... 151 Result Table... 151 Troubleshooting... 151 Exp. 4.4 Pulsed Field Gel Electrophoresis (PFGE) Analysis... 152 Introduction... 152 Principle... 153 Reagents Required and Their Role... 155 Procedure... 156 Observation... 157 Result Table... 157 Troubleshooting... 158 Precautions... 158 Exp. 4.5 Multiplex PCR for Rapid Characterization of Bacteria... 161 Introduction... 161 Principle... 162
xii Contents Reagents Required and Their Role... 162 Procedure... 164 Observation... 164 Result Table... 164 Troubleshooting... 165 Precautions... 165 Exp. 4.6 ERIC and REP-PCR Fingerprinting Techniques... 166 Introduction... 166 Principle... 167 Reagents Required and Their Role... 168 Procedure... 170 Observation... 171 Result Table... 171 Troubleshooting... 172 Precautions... 172 5 Computer-Aided Study of Molecular Microbiology... 175 Exp. 5.1 Analysis of Gene Sequences... 175 Introduction... 175 Example of Tools for Sequence Analysis... 175 Principle... 176 Procedure... 176 Exp. 5.2 Submission of Sequences to GenBank... 182 Introduction... 182 Principle... 183 Procedure... 183 Exp. 5.3 Phylogenetic Trees... 189 Introduction... 189 Reading Trees... 190 Phylogenetic Tree Software... 190 Principle... 190 Procedure... 192 Exp. 5.4 Primer Design... 197 Introduction... 197 Primer Designing Using Software... 198 Guidelines for Primer Design... 199 Procedure for Using NETPRIMER Software for Primer Designing... 199 6 Application of Molecular Microbiology... 203 Exp. 6.1 Biofilm Formation in Glass Tubes... 203 Introduction... 203 Principle... 204 Reagents Required and Their Role... 205 Procedure... 205 Observation... 206 Result Table... 206 Troubleshooting... 206
Contents xiii Precaution... 207 Exp. 6.2 Screening of Biofilm Formation in Micro-Titre Plates... 208 Introduction... 208 Principle... 209 Reagents Required and Their Role... 210 Procedure... 210 Observation... 211 Result Table... 211 Troubleshooting... 211 Precaution... 212 Exp. 6.3 Confocal Laser Scanning Microscopy for Biofilm Analysis... 214 Introduction... 214 Principle... 214 Reagents Required and Their Role... 216 Biofilm-Forming Bacteria... 216 Protocol... 217 Observation... 217 Observation Table... 217 Precautions... 218 Troubleshooting... 218 Exp. 6.4 Fluorescence Microscopy of Bacterial Biofilm and Image Analysis... 219 Introduction... 219 Principle... 220 Reagents Required and Their Role... 220 Protocol... 221 Observation Table... 221 Precautions... 224 Exp. 6.5 Screening for Biosurfactants... 225 Introduction... 225 Principle... 226 Reagents Required and Their Role... 227 Procedure... 227 Observation... 228 Result Table... 228 Exp. 6.6 Spectrophotometric Analysis of Bioremediation of Polycyclic Aromatic Hydrocarbons by Bacteria... 229 Introduction... 229 Principle... 229 Reagents Required and Their Role... 230 Procedure... 230 Observation... 231 Observation Table... 231 Precautions... 231 Exp. 6.7 H 2 S Assay to Screen Metal-Accumulating Bacteria... 232
xiv Contents Introduction... 232 Principle... 233 Reagents Required and Their Role... 234 Procedure... 234 Observation... 234 Result Table... 235 Troubleshooting... 235 Precautions... 235 References... 237 Further Readings... 239
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