Table Of ContentMicrobial Biotechnology- A Laboratory
Manual for Bacterial Systems
Surajit Das • Hirak Ranjan Dash
Microbial
Biotechnology- A
Laboratory Manual
for Bacterial Systems
Surajit Das Hirak Ranjan Dash
Department of Life Science Department of Life Science
National Institute of Technology National Institute of Technology
Rourkela Rourkela
Odisha Odisha
India India
ISBN 978-81-322-2094-7 ISBN 978-81-322-2095-4 (eBook)
DOI 10.1007/978-81-322-2095-4
Springer New Delhi Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014955535
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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.
v
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!
Rourkela, Odisha, India Surajit Das
Hirak R. Dash
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
