Contents. 1 Basic Molecular Microbiology of Bacteria... 1 Exp. 1.1 Isolation of Genomic DNA Introduction Principle...

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1 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 Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshootings Precautions Exp. 1.4 Isolation of Total RNA from Bacteria Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshootings Precautions Exp. 1.5 Amplification of 16S rrna Gene vii

2 viii Contents Introduction Principle Reagents Required and Their Role Procedure Observation Troubleshootings Precautions Exp. 1.6 To Perform Agarose Gel Electrophoresis Introduction Principle Reagents Required and Their Role Procedure Observation Troubleshootings Precautions Cloning and Transformation Exp. 2.1 Preparation of Competent Cells and Heat-Shock Transformation Introduction Principle Reagents Required and Their Role Procedure Observation Troubleshooting Precautions Exp. 2.2 Electroporation Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions Exp. 2.3 Restriction Digestion and Ligation Introduction Principle Reagents Required and Their Role Procedure Observation Troubleshooting Precaution Exp. 2.4 Selection of a Suitable Vector System for Cloning Different Types of Cloning Vectors Criteria for Choosing a Suitable Cloning Vector Conclusion Exp. 2.5 Confirmation of Transformation by Blue-White Selection... 62

3 Contents ix Introduction Principle Reagents Required and Their Role IPTG Antibiotics pbluescript Transformation Reaction Product Procedure Observation Troubleshooting Precautions Exp. 2.6 Confirmation of Cloning by PCR Introduction Principle Reagents Required and Their Role Procedure Observation Troubleshooting Precautions Advanced Molecular Microbiology Techniques Exp Synthesis of cdna Introduction Principle Reagents Required and Their Role Procedure Observation Trouble-Shootings Precautions Exp Gene Expression Analysis by qrt-pcr Introduction Principle Reagents Required and Their Role Procedure Observation Trouble-Shootings Precautions Exp Gene Expression Analysis Using Reporter Gene Assay Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Precaution Trouble-Shootings... 89

4 x Contents Exp Semi-quantitative Gene Expression Analysis Introduction Principle Reagents Required and Their Role Procedure Observation Observation Table Trouble-Shootings Precautions Exp Northern Blotting Introduction Principle Reagents Required and Their Role Procedure Observation Trouble-Shootings Precautions Exp Isolation of Metagenomic DNA Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Trouble-Shootings Precautions Exp Plasmid Curing from Bacterial Cell Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Trouble-Shootings Precautions Exp Conjugation in Bacteria Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Trouble-Shootings Precaution Exp Transduction in Bacteria Introduction Principle Reagents Required and Their Role

5 Contents xi Procedure Observation Result Table Trouble-Shootings Precaution Molecular Microbial Diversity Exp. 4.1 Plasmid Profile Analysis Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions Exp. 4.2 Amplified Ribosomal DNA Restriction Analysis to Study Bacterial Relatedness Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions Exp. 4.3 Denaturing Gradient Gel Electrophoresis (DGGE) Analysis to Study Metagenomic Bacterial Diversity Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Exp. 4.4 Pulsed Field Gel Electrophoresis (PFGE) Analysis Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions Exp. 4.5 Multiplex PCR for Rapid Characterization of Bacteria Introduction Principle

6 xii Contents Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions Exp. 4.6 ERIC and REP-PCR Fingerprinting Techniques Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions Computer-Aided Study of Molecular Microbiology Exp. 5.1 Analysis of Gene Sequences Introduction Example of Tools for Sequence Analysis Principle Procedure Exp. 5.2 Submission of Sequences to GenBank Introduction Principle Procedure Exp. 5.3 Phylogenetic Trees Introduction Reading Trees Phylogenetic Tree Software Principle Procedure Exp. 5.4 Primer Design Introduction Primer Designing Using Software Guidelines for Primer Design Procedure for Using NETPRIMER Software for Primer Designing Application of Molecular Microbiology Exp. 6.1 Biofilm Formation in Glass Tubes Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting

7 Contents xiii Precaution Exp. 6.2 Screening of Biofilm Formation in Micro-Titre Plates Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precaution Exp. 6.3 Confocal Laser Scanning Microscopy for Biofilm Analysis Introduction Principle Reagents Required and Their Role Biofilm-Forming Bacteria Protocol Observation Observation Table Precautions Troubleshooting Exp. 6.4 Fluorescence Microscopy of Bacterial Biofilm and Image Analysis Introduction Principle Reagents Required and Their Role Protocol Observation Table Precautions Exp. 6.5 Screening for Biosurfactants Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Exp. 6.6 Spectrophotometric Analysis of Bioremediation of Polycyclic Aromatic Hydrocarbons by Bacteria Introduction Principle Reagents Required and Their Role Procedure Observation Observation Table Precautions Exp. 6.7 H 2 S Assay to Screen Metal-Accumulating Bacteria

8 xiv Contents Introduction Principle Reagents Required and Their Role Procedure Observation Result Table Troubleshooting Precautions References Further Readings

Contents... vii. List of Figures... xii. List of Tables... xiv. Abbreviatons... xv. Summary... xvii. 1. Introduction In vitro evolution...

Contents... vii. List of Figures... xii. List of Tables... xiv. Abbreviatons... xv. Summary... xvii. 1. Introduction In vitro evolution... vii Contents Contents... vii List of Figures... xii List of Tables... xiv Abbreviatons... xv Summary... xvii 1. Introduction...1 1.1 In vitro evolution... 1 1.2 Phage Display Technology... 3 1.3 Cell surface