Microbial Biotechnology- A Laboratory Manual for Bacterial Systems

Surajit Das • Hirak Ranjan Dash

Microbial Biotechnology- A Laboratory Manual for Bacterial Systems

ISBN 978-81-322-2094-7 ISBN 978-81-322-2095-4 (eBook)DOI 10.1007/978-81-322-2095-4Springer New Delhi Heidelberg New York Dordrecht London

Library of Congress Control Number: 2014955535

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Surajit DasDepartment of Life ScienceNational Institute of TechnologyRourkelaOdisha India

Hirak Ranjan DashDepartment of Life ScienceNational Institute of TechnologyRourkelaOdisha India

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Preface

Though tiny in size, bacteria impart many useful applications for the sustain-able maintenance of the ecosystem on earth. On the evolutionary lineage, they are the first to appear and had plenty of time to adapt in the environmen-tal conditions, subsequently giving rise to numerous descendant forms. They are omnipresent in huge number and their diversity is extended from hydro-thermal vents to the cold seeps. These tiny, one-celled creatures carry out many useful functions and with the advancement of science, they have been explored greatly for use in food industry, agricultural industry, clinical sec-tors and many others. Biotechnological industries utilise bacterial cells for the production of biological substances that are useful for human existence including foods, medicines, hormones, enzymes, proteins and nucleic acids. Despite huge benefits human beings gain out of these microscopic organisms, less attention has been paid to study these tiny creatures. Though the research on bacterial entities has gained momentum, it is estimated that only about 1 % of the microorganisms have been discovered so far. However, rapid advances in molecular biology have revolutionised the study of bacteria in the envi-ronment. It has provided new insights regarding their composition, phylog-eny and physiology. New developments in biotechnology and environmental microbiology signify that microbiology will continue to be an exciting and emerging field of study in the future.

The study of bacteria dates back to 1900 AD and substantial advancement on the methodology and practices used for their study has been occurred. There are many textbooks, research and review articles dealing with state-of-art of various aspects of molecular biology of microorganisms. However, the users usually get lost in initiating an experiment due to lack of suitable easy protocols. In this regard, an assorted laboratory manual not only to motivate the researchers and students but also to enhance the acquisition of scientific knowledge as well as the scientific aptitude is the need of the hour. This laboratory manual ‘Microbial biotechnology—a laboratory manual for bac-terial systems’ is an attempt to overcome the inherent cumbersome practices that are followed in most of the laboratories. Every effort has been made to present the protocols in a very simpler form for easy understanding of the undergraduates, graduates, postgraduates, doctoral students, active scientists and researchers. Additionally, most of the universities providing undergradu-ate and postgraduate courses in microbiology and biotechnology, can use for their laboratory experiments.

vi Preface

There is a considerable difference between a researcher and a technician. The technician can add the appropriate reagents to obtain the suitable result. However, the researcher should focus on ‘how’ and ‘why’. Blindly follow-ing a protocol without knowing the principle and role of reagents will not be useful in a long run. Thus, an attempt has been made to make the novice students familiar with the principle of the each experimental setup and active role of each reagent to be used in each experiment. Thus, it will be help-ful for the readers to modify the protocols as well as the reagents as per their requirement. The illustrative description of each experiment will be of great use in easy understanding of the readers, irrespective of their qualifi-cation and research expertise. Some specific experiments in the advanced field of environmental microbiology have been included in the last part of the manual which will increase the awareness among the students regarding the vast application of these tiny microorganisms for the sustainability of the ecosystem.

We have tried our best to incorporate all our experience and expertise to come out in the form of this manual. Throughout the writing process of this manual we have faced lots of problems and hurdles. All have been overcome due to God’s grace, self-belief and people surrounding to us. We are highly thankful to each and every one for their support and encouragement in this process. We hope this manual will be of great use for the readers in their aca-demic and research career. Wishing all the very best to the readers and their experiments!

Surajit DasHirak R. Dash

Rourkela, Odisha, India

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Contents

1 Basic Molecular Microbiology of Bacteria ................................. 1Exp. 1.1 Isolation of Genomic DNA .............................................. 1Introduction ..................................................................................... 1Principle .......................................................................................... 1Reagents Required and Their Role ................................................ 2Procedure ........................................................................................ 3Observation ..................................................................................... 4Result Table .................................................................................... 4Troubleshootings ............................................................................. 4Precautions ...................................................................................... 4Exp. 1.2 Preparation of Bacterial Lysates ..................................... 5Introduction ..................................................................................... 5Principle .......................................................................................... 6Procedure ........................................................................................ 7Observation ..................................................................................... 9Result Table .................................................................................... 9Troubleshootings ............................................................................. 9Precautions ...................................................................................... 9Exp. 1.3 Isolation of Plasmids ...................................................... 12Introduction ................................................................................... 12Principle ........................................................................................ 13Reagents Required and Their Role .............................................. 13Procedure ...................................................................................... 15Observation ................................................................................... 15Result Table .................................................................................. 16Troubleshootings ........................................................................... 16Precautions .................................................................................... 16Exp. 1.4 Isolation of Total RNA from Bacteria ........................... 17Introduction ................................................................................... 17Principle ........................................................................................ 18Reagents Required and Their Role .............................................. 19Procedure ...................................................................................... 20Observation ................................................................................... 20Result Table .................................................................................. 21Troubleshootings ........................................................................... 21Precautions .................................................................................... 21Exp. 1.5 Amplification of 16S rRNA Gene ................................. 22

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Introduction ................................................................................... 22Principle ........................................................................................ 23Reagents Required and Their Role .............................................. 25Procedure ...................................................................................... 26Observation ................................................................................... 27Troubleshootings ........................................................................... 28Precautions .................................................................................... 28Exp. 1.6 To Perform Agarose Gel Electrophoresis ...................... 29Introduction ................................................................................... 29Principle ........................................................................................ 30Reagents Required and Their Role .............................................. 31Procedure ...................................................................................... 32Observation ................................................................................... 33Troubleshootings ........................................................................... 33Precautions .................................................................................... 34

2 Cloning and Transformation ...................................................... 35Exp. 2.1 Preparation of Competent Cells and Heat-Shock Transformation .............................................................................. 35Introduction ................................................................................... 35Principle ........................................................................................ 35Reagents Required and Their Role .............................................. 37Procedure ...................................................................................... 38Observation ................................................................................... 39Troubleshooting ............................................................................ 39Precautions .................................................................................... 39Exp. 2.2 Electroporation ............................................................... 41Introduction ................................................................................... 41Principle ........................................................................................ 42Reagents Required and Their Role .............................................. 43Procedure ...................................................................................... 43Observation ................................................................................... 44Result Table .................................................................................. 45Troubleshooting ............................................................................ 45Precautions .................................................................................... 45Exp. 2.3 Restriction Digestion and Ligation ............................... 46Introduction ................................................................................... 46Principle ........................................................................................ 47Reagents Required and Their Role .............................................. 50Procedure ...................................................................................... 51Observation ................................................................................... 52Troubleshooting ............................................................................ 52Precaution ..................................................................................... 53Exp. 2.4 Selection of a Suitable Vector System for Cloning ...... 54Different Types of Cloning Vectors ............................................. 55Criteria for Choosing a Suitable Cloning Vector ........................ 60Conclusion .................................................................................... 62Exp. 2.5 Confirmation of Transformation by Blue-White Selection .................................................................... 62

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Introduction ................................................................................... 62Principle ........................................................................................ 63Reagents Required and Their Role .............................................. 64IPTG .............................................................................................. 64Antibiotics ..................................................................................... 65pBluescript .................................................................................... 65Transformation Reaction Product ................................................ 65Procedure ...................................................................................... 65Observation ................................................................................... 65Troubleshooting ............................................................................ 66Precautions .................................................................................... 66Exp. 2.6 Confirmation of Cloning by PCR ................................. 67Introduction ................................................................................... 67Principle ........................................................................................ 68Reagents Required and Their Role .............................................. 68Procedure ...................................................................................... 70Observation ................................................................................... 70Troubleshooting ............................................................................ 71Precautions .................................................................................... 71

3 Advanced Molecular Microbiology Techniques ....................... 73Exp. 3.1. Synthesis of cDNA ........................................................ 73Introduction ................................................................................... 73Principle ........................................................................................ 73Reagents Required and Their Role .............................................. 75Procedure ...................................................................................... 76Observation ................................................................................... 77Trouble-Shootings ......................................................................... 78Precautions ................................................................................... 78Exp. 3.2. Gene Expression Analysis by qRT-PCR ...................... 79Introduction ................................................................................... 79Principle ........................................................................................ 80Reagents Required and Their Role .............................................. 82Procedure ...................................................................................... 83Observation ................................................................................... 84Trouble-Shootings ......................................................................... 85Precautions .................................................................................... 85Exp. 3.3. Gene Expression Analysis Using Reporter Gene Assay .................................................................... 86Introduction ................................................................................... 86Principle ........................................................................................ 87Reagents Required and Their Role .............................................. 87Procedure ...................................................................................... 88Observation ................................................................................... 89Result Table .................................................................................. 89Precaution ..................................................................................... 89Trouble-Shootings ......................................................................... 89

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Exp. 3.4. Semi-quantitative Gene Expression Analysis .............. 90Introduction ................................................................................... 90Principle ........................................................................................ 91Reagents Required and Their Role .............................................. 92Procedure ...................................................................................... 94Observation ................................................................................... 94Observation Table ......................................................................... 95Trouble-Shootings ......................................................................... 96Precautions .................................................................................... 96Exp. 3.5. Northern Blotting .......................................................... 97Introduction ................................................................................... 97Principle ........................................................................................ 98Reagents Required and Their Role .............................................. 99Procedure ...................................................................................... 100Observation ................................................................................... 102Trouble-Shootings ......................................................................... 102Precautions .................................................................................... 103Exp. 3.6. Isolation of Metagenomic DNA ................................... 104Introduction ................................................................................... 104Principle ........................................................................................ 105Reagents Required and Their Role .............................................. 106Procedure ...................................................................................... 107Observation ................................................................................... 108Result Table .................................................................................. 108Trouble-Shootings ......................................................................... 108Precautions .................................................................................... 109Exp. 3.7. Plasmid Curing from Bacterial Cell ............................. 109Introduction ................................................................................... 109Principle ......................................................................................... 110Reagents Required and Their Role ............................................... 111Procedure ...................................................................................... 112Observation ................................................................................... 112Result Table .................................................................................. 112Trouble-Shootings ....................................................................... 113Precautions .................................................................................. 113Exp. 3.8. Conjugation in Bacteria ............................................... 114Introduction .................................................................................. 114Principle ....................................................................................... 114Reagents Required and Their Role ............................................ 115Procedure ..................................................................................... 116Observation .................................................................................. 116Result Table ................................................................................. 117Trouble-Shootings ........................................................................ 117Precaution .................................................................................... 117Exp. 3.9. Transduction in Bacteria .............................................. 118Introduction .................................................................................. 118Principle ...................................................................................... 119Reagents Required and Their Role ............................................ 120

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Procedure .................................................................................... 121Observation ................................................................................. 122Result Table ................................................................................ 122Trouble-Shootings ....................................................................... 122Precaution ................................................................................... 122

4 Molecular Microbial Diversity ................................................. 125Exp. 4.1 Plasmid Profile Analysis .............................................. 125Introduction ................................................................................. 125Principle ...................................................................................... 125Reagents Required and Their Role ............................................ 126Procedure .................................................................................... 128Observation ................................................................................. 129Result Table ................................................................................ 129Troubleshooting .......................................................................... 132Precautions .................................................................................. 132Exp. 4.2 Amplified Ribosomal DNA Restriction Analysis to Study Bacterial Relatedness ................................... 134Introduction ................................................................................. 134Principle ...................................................................................... 135Reagents Required and Their Role ............................................ 136Procedure .................................................................................... 138Observation ................................................................................. 139Result Table ................................................................................ 142Troubleshooting .......................................................................... 142Precautions .................................................................................. 143Exp. 4.3 Denaturing Gradient Gel Electrophoresis (DGGE) Analysis to Study Metagenomic Bacterial Diversity ................ 144Introduction ................................................................................. 144Principle ...................................................................................... 145Reagents Required and Their Role ............................................ 146Procedure .................................................................................... 147Observation ................................................................................. 151Result Table ................................................................................ 151Troubleshooting .......................................................................... 151Exp. 4.4 Pulsed Field Gel Electrophoresis (PFGE) Analysis .... 152Introduction ................................................................................. 152Principle ...................................................................................... 153Reagents Required and Their Role ............................................ 155Procedure .................................................................................... 156Observation ................................................................................. 157Result Table ................................................................................ 157Troubleshooting .......................................................................... 158Precautions .................................................................................. 158Exp. 4.5 Multiplex PCR for Rapid Characterization of Bacteria ................................................................................... 161Introduction ................................................................................. 161Principle ...................................................................................... 162

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Reagents Required and Their Role ............................................ 162Procedure .................................................................................... 164Observation ................................................................................. 164Result Table ................................................................................ 164Troubleshooting .......................................................................... 165Precautions .................................................................................. 165Exp. 4.6 ERIC and REP-PCR Fingerprinting Techniques ........ 166Introduction ................................................................................. 166Principle ...................................................................................... 167Reagents Required and Their Role ............................................ 168Procedure .................................................................................... 170Observation ................................................................................. 171Result Table ................................................................................ 171Troubleshooting .......................................................................... 172Precautions .................................................................................. 172

5 Computer-Aided Study of Molecular Microbiology .............. 175Exp. 5.1 Analysis of Gene Sequences ........................................ 175Introduction ................................................................................. 175Example of Tools for Sequence Analysis .................................. 175Principle ...................................................................................... 176Procedure .................................................................................... 176Exp. 5.2 Submission of Sequences to GenBank ....................... 182Introduction ................................................................................. 182Principle ...................................................................................... 183Procedure .................................................................................... 183Exp. 5.3 Phylogenetic Trees ....................................................... 189Introduction ................................................................................. 189Reading Trees ............................................................................. 190Phylogenetic Tree Software ........................................................ 190Principle ...................................................................................... 190Procedure .................................................................................... 192Exp. 5.4 Primer Design .............................................................. 197Introduction ................................................................................. 197Primer Designing Using Software ............................................. 198Guidelines for Primer Design .................................................... 199Procedure for Using NETPRIMER Software for Primer Designing .................................................................. 199

6 Application of Molecular Microbiology .................................. 203Exp. 6.1 Biofilm Formation in Glass Tubes ............................... 203Introduction ................................................................................. 203Principle ...................................................................................... 204Reagents Required and Their Role ............................................ 205Procedure .................................................................................... 205Observation ................................................................................. 206Result Table ................................................................................ 206Troubleshooting .......................................................................... 206

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Precaution ................................................................................... 207Exp. 6.2 Screening of Biofilm Formation in Micro-Titre Plates ....................................................................... 208Introduction ................................................................................. 208Principle ...................................................................................... 209Reagents Required and Their Role ............................................ 210Procedure .................................................................................... 210Observation .................................................................................. 211Result Table ................................................................................. 211Troubleshooting ........................................................................... 211Precaution ................................................................................... 212Exp. 6.3 Confocal Laser Scanning Microscopy for Biofilm Analysis ................................................................... 214Introduction ................................................................................. 214Principle ...................................................................................... 214Reagents Required and Their Role ............................................ 216Biofilm-Forming Bacteria .......................................................... 216Protocol ........................................................................................ 217Observation .................................................................................. 217Observation Table ........................................................................ 217Precautions .................................................................................. 218Troubleshooting .......................................................................... 218Exp. 6.4 Fluorescence Microscopy of Bacterial Biofilm and Image Analysis ....................................................... 219Introduction ................................................................................. 219Principle ...................................................................................... 220Reagents Required and Their Role ............................................ 220Protocol ....................................................................................... 221Observation Table ....................................................................... 221Precautions .................................................................................. 224Exp. 6.5 Screening for Biosurfactants ....................................... 225Introduction ................................................................................. 225Principle ...................................................................................... 226Reagents Required and Their Role ............................................ 227Procedure .................................................................................... 227Observation ................................................................................. 228Result Table ................................................................................ 228Exp. 6.6 Spectrophotometric Analysis of Bioremediation of Polycyclic Aromatic Hydrocarbons by Bacteria ................... 229Introduction ................................................................................. 229Principle ...................................................................................... 229Reagents Required and Their Role ............................................ 230Procedure .................................................................................... 230Observation ................................................................................. 231Observation Table ....................................................................... 231Precautions .................................................................................. 231Exp. 6.7 H2S Assay to Screen Metal-Accumulating Bacteria ....................................................................................... 232

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Introduction ................................................................................. 232Principle ...................................................................................... 233Reagents Required and Their Role ............................................ 234Procedure .................................................................................... 234Observation ................................................................................. 234Result Table ................................................................................ 235Troubleshooting .......................................................................... 235Precautions .................................................................................. 235

References ........................................................................................ 237

Further Readings ............................................................................ 239

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About the Authors

Surajit Das is an Assistant Professor at the Department of Life Science, National Institute of Technology, Rourkela, Orissa, India since 2009. Ear-lier he served at Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, India. He received his Ph.D. in Marine Biology (Microbiol-ogy) from Centre of Advanced Study in Marine Biology, Annamalai Uni-versity, Tamil Nadu, India. He has been the awardee of Endeavour Research Fellowship of Australian Government for carrying out Postdoctoral research at University of Tasmania on marine microbial technology. He has multiple research interests with core research program on marine microbiology. He is currently conducting research as the group leader of Laboratory of Environ-mental Microbiology and Ecology (LEnME) on biofilm based bioremedia-tion of PAHs and heavy metals by marine bacteria, metagenomic approach for drug discovery from marine microorganisms, nanoparticle-based drug delivery and bioremediation; and the metagenomic approach for exploring the diversity of catabolic gene and immunoglobulins in the Indian Major Carps, with the help of research grants from the Department of Biotechnol-ogy (DBT), Ministry of Science and Technology and the Indian Council of Agricultural Research (ICAR), Government of India. Recognizing his work, National Environmental Science Academy, New Delhi had conferred 2007 Junior Scientist of the year award on marine microbial diversity. He is the recipient of Young Scientist Award in Environmental Microbiology from Association of Microbiologists of India in 2009. Dr. Das is also the recipi-ent of Ramasamy Padayatchiar Endowment Merit Award given by Govern-ment of Tamil Nadu for the year 2002-2003 from Annamalai University. He is the member of IUCN Commission of Ecosystem Management (CEM), South Asia and life member of the Association of Microbiologists of India, Indian Science Congress Association, National Academy of Biological Sci-ences and National Environmental Science Academy, New Delhi. He is also the member of the International Association for Ecology. He is the reviewer of many scientific journals published by reputed publishers. He has writ-ten three books and authored more than 40 research publications in leading national and international journals on different aspects of microbiology.

xvi About the Authors

Hirak Ranjan Dash is a Senior Research Fellow at Laboratory of Environ-mental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, Odisha, India. He did his M. Sc. Microbiology (2010) from Orissa University of Agriculture and Technol-ogy, Bhubaneswar, Odisha, India. Currently, he is continuing his research on diversity and genetic aspects of mercury resistant marine bacteria for enhanced bioremediation of mercury. He has also worked in the field of anti-biotic resistance and genotyping of pathogenic Vibrio and Staphylococcus spp. During his research work, he has isolated many potent mercury resistant marine bacteria from Bay of Bengal, Odisha and utilised in mercury biore-mediation. A number of microbiological technique has also been developed by him for monitoring the level of mercury pollution in the marine environ-ment. A novel mechanism of mercury resistance i.e. by intracellular biosorp-tion was reported by him in the marine bacterial isolates. He has constructed transgenic marine bacteria possessing both mercury biosorption and volatil-ization capability for utilization in mercury bioremediation. He has published 14 research papers, 7 book chapters and 10 conference proceedings in his credit.