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 Prep
TM

Module
Notes
12
CSIR - NET

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Applied
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Biology
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Dr. Aditya Arya | Dr. Amit Kumar

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®
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Module 12

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Applied Biology
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Drawing Pin Publishing

New Delhi, India.
ii
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Module 12

Applied Biology G
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Editors
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Dr. Aditya Arya, Ph.D.

Dr. Amit Kumar, Ph.D.

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Drawing Pin Publishing

New Delhi, India.

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Drawing Pin PublishingTM, New Delhi, India

Prep-NoteTM CSIR-NET Module 12 - Applied Biology, 1st Edition

Pre-print : 1st Jan 2022, First Edition: 29 April 2022.

ASIN : B07B3SWP6C

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Copyright © 2022. Drawing Pin Publishing. All Rights Reserved.

All rights reserved. No part of this publication including text, tables, and illustrations
may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any other storage and retrieval
system, without the prior written permission of the publisher. All the copyright-

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related queries may be directly sent to the publisher at
[email protected].
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Disclaimer

Although utmost care has been taken to avoid any errors in the preparation of
answers, however in case of any discrepancies or loss of any kind due to incorrect
answers or solutions, authors or publishers shall not be responsible. The sources of
information are cited in the online free supplement.
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Value Creativity and Hard Work

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At drawing pin publishing, we value the efforts of all team members and therefore
provide you a glimpse of efforts that have been put in to achieve this book in the form
of human hours and plagiarism declaration. We, therefore, request you to value hard
work and insist not to copy or discredit. Discrediting, not only causes a financial loss to
the publisher but also a moral loss to authors and community. Value originality and
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creativity by respecting copyright. Drawing pin publishing strictly follows the anti-
plagiarism policy as per international guidelines and acknowledges the hard work,
originality, and creativity.
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 Human hours involved ~ 6500 Hh

Plagiarism declaration

It is certified by the authors that anti-plagiarism check was donebefore publishing this
book and the content was found to be > 70 % unique.

Series Editor: Dr. Aditya Arya (Editor-in-Chief)

Cover design and illustrations: Dr. Aditya Arya (Series Editor and Editor-in-Chief)

Typesetting Editor: Mr. Ram Kumar

Printed at
New Delhi, India.

iv
 Preface

With our preceeding Module 13 for techniques which received a huge appreciation and acclaim by readers,
we are glad to introduce this CSIR-NET preparatory module 12, which has been prepared by the extensive
efforts of more than three years and about 6500 human hours. Considering the applied biology as one of
the most critical factors for a naive researcher, the importance of this module is well- justified. Although
this module has been prepared with a focus of one of well-known competitive examination for research
fellowship and lectureship named CSIR-NET, the module will also be highly useful for preparation of other
competitive fellowship examinations (ICMR, ICAR, DBT, GRE, etc.) and interviews for various M.Sc and

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Ph.D. positions. This book will also be a good companion for a beginner in research and lab technicians.
We observed the problems faced by research aspirants in finding a suitable text for preparing the methods
in biology and thus begun this work long back in 2018. We also understand that the scope and extent of
methods in biology are enormous and we cannot limit all the methods in one small book. However, we
have tried to cover most of the techniques to utmost clarity and depth in this module. This module
comprises of eight chapters subdivided from chapter A through chapter H. Chapter A is quite extensive

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and includes basics of rMicrobial fermentation and various biochemical aspects of microbial metabolism
Other chapters include plant and animal cell culture (Chapter B), genetically modified organisms (Chapter
C), Genomics (Chapter D), Biodiversity (Chapter E), Breeding in plants and animals and marker assisted
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selection (Chapter F), Bioremediation (Chapter G) and Biosesnors (Chapter H).
In order to improve the clarity of concepts, a number of simplified illustrations have been added to the
text. Also, we have made enough emphasis on analysis of data and graphical presentation of data obtained
from various techniques, which is the prime requirement of various fellowship exams as well as interview
questions. A number of brain teaser questions can also be found flowing with the text. These questions
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also contain a hint or suitable answers. However, due to the elaboration of text, we have refrained from
including the previous year questions in the main text, rather included as a appendix of this book.
The strength of this book primarily lies on the expertise of our editors, Dr. Aditya Arya and Dr. Amit Kumar,
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who not only have a very extensive research experience but also have been trained on most of the
techniques in prestigious institutes. The editors have also published a number of research papers which
was a critical factor in maintaining the quality of this book for the level of emerging researchers. The
passion for writing and contributing the learnings to the budding scientists was one of the driving force
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behind this book. Some “high-value notes” published by us shall also augment the preparation of methods.
Additionally, charts, interpret cards shall also be made available to support the understanding of various
methods in biology.
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We wish our readers and budding researchers good luck and welcome suggestions, critiques for the
improvement of this book.

Executive Editor
Drawing Pin Publishing.

v
 About The Editors
Dr. Aditya Arya, Ph.D.
Dr. Arya is a Ph.D. in the area of nanomedicine from defense research and development organization.
Earlier he completed his Master’s degree in Biochemistry from Madurai Kamaraj University. He has re-
search experience of more than a decade with a proven track record. He has published more than 35+
research papers in peer-reviewed journals which have attained 500+ citations. He has been trained at
some of the most prestigious institutions across the globe, such as Wellcome Trust Sanger Institute,
Cambridge, IBRO, Paris, and EMBL, Heidelberg and holds an active membership of several research soci-
eties in redox biology and nanomedicine. Besides this, he is also an academician and authored several
book chapters in international books and some of the successful books for graduate and postgrads, Con-

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cise Biochemistry, Understanding Enzymes to name a few. Dr. Arya has extensive experience in several
techniques such as flow cytometry, mass spectrometry, electron microscopy, x-ray diffraction, microarray,
radiolabelling, behavioral studies, and gene cloning. He is also a public speaker and science education
researcher, as he has developed a number of interesting concepts and analogies in various life sciences
subjects. He has delivered a number of scientific lectures at national and international platforms.

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Dr. Amit Kumar, Ph.D.
Dr. Amit is a Ph.D. in the area of Marine Molecular Ecology from Stazione Zoologica Anton Dohrn, Naples,
Italy. He has published 15+ research papers and book chapters in peer-reviewed international journals/
books. He holds expertise in oxidative and nitrosative stress enzymology of marine plants. Dr. Amit is also
the recipient of prestigious international research fellowships and grants in the domain of Marine Ecology.
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He was trained in various reputed research laboratories in Europe, including Verona University, Antwerp
University, Roscoff Marine Station, and the University of Gothenburg. He has delivered several scientific
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talks at national and international platforms. He is an expert open water diver and participated in several
scientific cruise expeditions. He has expertise in several field biology methods such as population estima-
tion, habitat characterization, DNA sequencing, expression analysis, microbial taxonomy, etc. His current
research interest is in the domain of microbial ecology of halophiles and the impact of environmental
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changes on the marine ecosystem.

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--------------------------------------------------------------------------------------------------------------
Free Online Resources
o Additional techniques (one page summary of more than 15
additonal techniques)
o Colour images of some of the figures of this module
o Cited text and further readings

Add-on Resources *
o Set of 8 posters, one for each chapter
o High value note on Agrobacterium mediated gene transfer
o Audio lectures of author (USB Stick)
* Add on resources are available at extra cost from the publisher.

viii
 Contents in Brief

Chpater A : Microbial Fermentation

Chpater B : Tissue Culture Methods (Plants and Animals)

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Chpater C: Transgenic Animals and Plants

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Chapter D: Genomics and Its Applications
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Chpater E: Bioresources and use of Biodiversity
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Chapter F: Breeding in Plants and Animals

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Chpater G: Bioremediation and Phytoremediation

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Chapter H: Biosensors
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Appnendix 1. CSIR-NET JRF Questions with Solutions

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 CHAPTER

Microbial
Fermentation A

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1. Introduction
Microbes represent some of the oldest living entities on the globe and therefore provide an additional evolu-

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tionary insight into the metabolic cascades of living beings. Unlike the biochemical pathways of higher organ-
isms particularly animals, microbes have varying sources of energy as well as diverse metabolic products. On
the basis of source of energy and carbon source microbes have been classified into various categories as
described in table A1. PA
Table A1. Classification of organisms by the energy and carbon source
Classification Energy source Carbon source Examples
Chemoautotrophs Chemical Inorganic Hydrogen-sulphur, iron, nitrogen, and carbo-monoxide bacteria
Chemoautotrophs Chemical Organic comp All animals, most fungi, protozoan, bacteria
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Photoautotrophs Light Inorganic All plants, algae, cyanobacteria, green and purple-S-bacteria
Photoheterotrophs Light Organic Green and purple non-sulphur bacteria, helicobacteria
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One of the characteristic metabolic phenomenon of microorganisms is fermentation. The use of fermenta-
tion, particularly for beverages, has existed since the Neolithic and has been documented dating from 7000–
6600 BCE in Jiahu, China, 6000 BCE in Georgia, 3150 BCE in ancient Egypt, 3000 BCE in Babylon, 2000 BCE in
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pre-Hispanic Mexico, and 1500 BC in Sudan. Fermented foods have a religious significance in Judaism and
Christianity. The Baltic god Rugutis was worshiped as the agent of fermentation. Louis Pasteur (1822–1895),
during the 1850s and 1860s, showed that fermentation is initiated by living organisms in a series of investiga-
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tions. In 1857, Pasteur showed that lactic acid fermentation is caused by living organisms. In 1860, he demon-
strated that bacteria cause souring in milk, a process formerly thought to be merely a chemical change, and
his work in identifying the role of microorganisms in food spoilage led to the process of pasteurization. In
1877, working to improve the French brewing industry, Pasteur published his famous paper on fermentation,
“Etudes sur la Bière”, which was translated into English in 1879 as “Studies on fermentation”. He defined
fermentation (incorrectly) as “Life without air”, but correctly showed that specific types of microorganisms
cause specific types of fermentations and specific end-products. In this chapter we will discuss about the basic
idea of fermentation, its types and reactions involved, followed by discussion on aspects of industrial fermen-
tation and production of metabolites at commercial level.

2. Defining Fermentation
For a larger group of naïve people, fermentation simply means the production of alcohol. If a food soured, one
might say it was ‘off’ or fermented. However fermentation is a much broader metabolic term. Informally, it
 Module 12: Applied Biology

as well as industrial alcohol. Yeasts are essentially aerobic organisms, but they can also grow as facultative anaer-
obes. The energy-yield under anaerobic conditions is much lower and hence the growth is slower with much lower
cell-yield. When grown with aeration, the cell-yield increases dramatically, but alcohol production falls. Thus, oxy-
gen inhibits fermentation. This is known as Pasteur-effect.

Homolactic Fermentation

Mixed Acid Fermentation

NAD+

O NADH
O Pyruvate
(2 mole) NADH NAD+
CH2 C C

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NAD+ CO2 NADH
OH
DG = -25.1 kJ mol-1 Malate OAA Pyruvate Lactate
NADH Py Corboxylase LDH
Lactate Dehydrogenase
NAD+ Pyruvate
OH formate
O Succinate lyase
Acetyl CoA

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CH2 C C NADH Formate
OH Lactic Acid NAD+
(2 mole)
H Ethanol Acetaldehyde CO2 +H2
EMP or ED Pathway Heterolactic
Fermentation
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Glucose
Acetate

O
Pyruvate
O
Alcohol Fermentation
CH2 C C
OH
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NAD+ G = ? kJ mol-1
CO2
NADH OH
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6-phosphogluconate Alcohol Dehydrogenase O

Phosphoketolase
NAD+ CH2 CH2 CH2 C
Pathway
CO2 H
NADH Ethanol NAD+ NADH
Acetaldehyde
Ribulose-5 Phosphate
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Alkaline Fermentation
NADH NAD+
Proteins
NAD+ Xylulose-5 Phosphate NADH

Pyruvate Acetyl CoA Acetaldehyde

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G-3-P Ac-3-P Amino acids

Phosphoketolase NADH
NADH
NAD+
NAD+
NH3+
Lactic Acid Ethanol

Fig A2. Outline of various biochemical pathways involved in fermentation process.

2.2.2 Homolactic Fermentation

Homolactic fermentation (producing only lactic acid) is the simplest type of fermentation. The pyruvate from glyco-
lysis undergoes a simple redox reaction, forming lactic acid. It is unique because it is the only respiration processes
to not produce a gas as a byproduct. Overall, one molecule of glucose (or any six-carbon sugar) is converted to two
molecules of lactic acid. It occurs in the muscles of animals when they need energy faster than the blood cannot
supply enough oxygen. It occurs in some kinds of bacteria and some fungi. Bacteria often converts lactose into lactic
acid in yogurt, giving it its sour taste. These lactic acid bacteria can carry out either homolactic fermentation, where

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 Module 12: Applied Biology

The oxygen solubility in water between 0 and 40oC is approximately. The biological oxygen demand (BOD) of a
culture depends on the substrate used in the fermentation, ranging from 0.6–0.8 (kg O2/kg dry weight) for the
partially oxidized glucose up to 5.0–5.6 for fully reduced methane. Assuming complete oxidation of the substrate for
biomass biosynthesis, the oxygen demand can be expressed as

BOD = {Oxygen for oxidation of substrate needed for 1kg biomass} – {Oxygen for oxidation of 1 kg biomass}

Furthermore, another term called oxygen uptake rate (OUR) is used to represent oxygen uptake during steady
state, it is equals the oxygen intake (or OTR, oxygen transfer rate) given by formula

OUR = rO2 = BOD × rX = BOD = × 5Øß × X = 3600 × kLa × (C * “ CL)(kg(m3 h)”1)

Where, kLa = volumetric oxygen mass transfer coefficient (s”1), OUR = oxygen uptake rate (kg O2 (m3 × h)”1, C* =

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oxygen solubility (kg m”3) and CL = oxygen concentration in medium (kg m”3).

----------------------------------------------------------------------------------------------------------------------------------
Brain Teaser

Q. What is Leibig’s law of the minimum and Shelford’s law of tolerance?

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Hint: Leibig Law of minimum states that total biomass of organism determined by nutrient present at lowest concentration. Shelford’s law of
tolerance states that above or below certain environmental limits, a microorganism will not grow, regardless of the nutrient supply.

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3.2 Growth Kinetics of Microbes

Microbial growth kinetics explains the relationship between the specific growth rate of a microbe and its substrate
concentration. Microbial growth kinetics largely depends on the laboratory culture conditions. In batch culture,
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microbial cell composition and its state change as a function of time and thus the rate of increase in biomass
concentration was monitored. The substrate such as nutrients (carbon and nitrogen sources), hormones and growth
factors influence the growth pattern of microbial and mammalian cells.
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Growth curve
Lag phase Log phase The rate of cell
Doubling
time (td)
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division equals
ln X/Xo

the rate of cell X

A period of death and hence td = slope = mmax
X0
adaptation no change
for the cells to in number
time
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their new
environment Death rate is
Death phase
higher than growth
Maximum growth (mm)
due to toxic
accumulation or
nutrient depletion
mm =
ln 2 0.693
=
td td
0 Time (days) 10
A period of
adaptation
for the cells to Log phase © 2022. Drawing Pin Publishing.
their new
environment

Fig A5. Typical kinetics of growth of a microbial culture

3.2.1 Phases of Microbial Growth

Microbial growth in culture media is characterized by three important phases viz, lag phase, log phase and station-
ary phase, the next phase after stationary is death phase. Lag Phase: The single cell inoculation into the liquid

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 Module 12: Applied Biology

Exercise
Q1. Explore the metabolic and biochemical similarities and differences between Lactic acid fermentation in Human
skeletal muscles and in the curdling of milk?

Q2. How is Bread manufactured? How many different types of microbes are used in standard bread manufacture?
can you contact any bakery to learn about the microbial product they use?

Q3. How Swiss cheese is different from conventional approach? Some special types of Swiss-cheese are produced
only in limited geographical regions and nowhere else in World, Why?

Q4. What is the differencebetween chemostat and turbidostat?

Q5. Comare and contrast growth-associated product formation, non-growth associted product fornation and mixed

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growth associated product formation and their kinetic equations.

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PA
E
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20
 CHAPTER

I- Vaccines and
Applications B

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1. Introduction
A vaccine is a biological preparation that establishes or improves immunity to a particular disease. Vaccines

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can be prophylactic (e.g. to prevent or ameliorate the effects of a future infection by any natural or “wild”
pathogen), or therapeutic (e.g. vaccines against cancer). The practice of immunisation dates back hundreds
of years. Buddhist monks drank snake venom to confer immunity to snake bite and variolation (smearing of a
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skin tear with cowpox to confer immunity to smallpox) was practiced in 17th century China. Edward Jenner is
considered the founder of vaccinology in the West in 1796, after he inoculated a 13 year-old-boy with vaccinia
virus (cowpox), and demonstrated immunity to smallpox. The term vaccine infact originated from vacca –
means cow which was also the reason why vaccine virus was named so. In 1798, the first smallpox vaccine
was developed. Over the 18th and 19th centuries, systematic implementation of mass smallpox immunisation
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culminated in its global eradication in 1979. Louis Pasteur’s experiments spearheaded the development of live
attenuated cholera vaccine in 1897 and inactivated anthrax vaccine in 1904.
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Africans started Origin of Origin of Meningitis C Pnemonococcal Development

using variolation term Vaccination term Vaccination vaccine conjugate of Covid-19
after Jenner's Work after Jenner's Work developed vaccine developed Vaccine
1706 1803 1962 1977 2000 2020
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1718 1885 1972 1981 2019

Variolation started Invention of Haemophilus Hepatitis- B Development

in Turkey Rabies Vaccine influenza vaccine Vaccine of Ebola
developed developed Vaccine

Fig B1. Timeline of vaccine development.

Plague vaccine was also invented in the late 19th Century. Between 1890 and 1950, bacterial vaccine develop-
ment proliferated, including the Bacillis-Calmette-Guerin (BCG) vaccination against tuberculosis, which is still
B1. Vaccines and their Applications

4.1 Recombinant Vector vaccines

Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens.
Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives
to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against
few protective antigens. There are a variety of expression systems with different advantages, allowing the produc-
tion of large quantities of proteins depending on the required characteristics. These vaccines are created by com-
bining the physiology of one micro-organism and the DNA of the other, immunity can be created against diseases
that have complex infection processes. In recent years a new type of vaccine, created from an infectious agent’s
DNA called DNA vaccination, has been developed. It works by insertion (and expression, triggering immune system
recognition) into human or animal cells, of viral or bacterial DNA. Some cells of the immune system that recognize
the proteins expressed will mount an attack against these proteins and cells expressing them. Because these cells

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live for a very long time, if the pathogen that normally expresses these proteins is encountered at a later time, they
will be attacked instantly by the immune system. Recombinant vaccines can be of various types depending upon the
molecules considered for insertion, most commonly they are categorised as recombinant protein vaccine, recombi-
nant viral vector vaccines and recombinant subunit vaccines.

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4.2 Recombinant Subunit Vaccine
Recombinant protein vaccines, also called recombinant subunit vaccines, are formulated using defined protein
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antigens that can be produced in heterologous expression systems. Recombinant protein subunit vaccines are
composed of at least 1 type of viral antigen that can be produced in heterologous expression systems. Recombinant
protein subunit vaccines are significantly more secure than live attenuated and inactivated/killed vaccines. In 1986,
the Recombivax HB vaccine for hepatitis B was approved for human use in several countries, the culmination of
research started by William Rutter, Pablo Valenzuela and colleagues in 1979 on the cloning of hepatitis B virus (HBV)
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antigens. It was the first vaccine to be produced using recombinant DNA technology and although it was only the
third recombinant product to be approved for clinical use (Fig B4).
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HBsAg
trpI
2m Transform yeast cells

EcoRI Ligation Transform yeast cells

HBsAg
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digest

ADH
Selection using Trp
trpI
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2m Culture on suitable free media

media
pMA56
yeast
vector ADH
Promoter

Lyse, spin
© 2022. Drawing Pin Publishing. and purify HBsAg

Fig B4. Design and development process of first recombinant subunit vaccine HBsAG

Some recombinant viral antigens can spontaneously assemble into virus-like particles (VLPs) which are multiprotein
structures without the incorporation of a viral genome. At present, some VLPs vaccines have been licensed and
commercialized. For example, 2 VLP-based vaccines produced in yeast, GlaxoSmithKline’s Engerix-B® (hepatitis B
virus, HBV) and Gardasil (human papillomavirus, HPV), are currently commercialized worldwide. A large num-
ber of VLP-based vaccine candidates are undergoing preclinical or clinical evaluations, such as influenza virus and
parvovirus VLPs.

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 Module 12: Applied Biology

4.3 Recombinant Vector Vaccine

Recombinant vector vaccines are live replicating viruses that are engineered to carry extra genes derived from a
pathogen—and these extra genes produce proteins against which we want to generate immunity. These vaccine
genomes may evolve to lose the extra genes during the process of manufacture of the vaccine or during replication
within an individual, and there is a concern that this evolution might severely limit the vaccine’s efficacy. The use of
attenuated vaccines is too risky for pathogens such as HIV, and a safer alternative is to develop a live, recombinant
vector vaccine where one or a few pathogen genes with immunogenic activity (proteins that elicit protective immu-
nity) are expressed from a benign virus vector. Viral vector-based vaccines present advantages over traditional
vaccines in that they can enhance a broad range of immunogenicity without an adjuvant and induce a robust
cytotoxic T lymphocyte (CTL) response to eliminate virus-infected cells. Several viral vectors have been common
used for developing vaccines such as adenovirus, adenoassociated virus, cytomegalovirus etc [A more detailed

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description on viral vectors is provided in chapter 12 E, gene therapy section]. Just like viral vectors, bacterial vector
based vaccines have also been tested. Compared to other types of vaccine carriers, bacteria-based antigen delivery
vectors exhibit multiple advantages such as their intrinsic infectious power, non-integrative properties and the ability
to regulate the amount and in vivo localization of antigen expression.

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4.4 DNA vaccines
A DNA vaccine (or genetic vaccine as it is also called) involves the direct injection of a naked DNA plasmid into
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muscle as a vaccine system with the ability to induce an immune response and protection after challenge is now
well established and used successfully. A DNA vaccine consists of a plasmid containing, one origin of replication of
Escherichia coli, for the amplification of the plasmid, a strong promoter, generally from cytomegalovirus, multiple
cloning sites, in which one can insert the gene to be expressed, and an antibiotic as selection marker.
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Typical DNA construct
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Cloning
Muscle site
Promoter
DNA (or RNA) Vaccine Enhancer
BGHpA
(Poly A site)
Ori Kanr
pDNA pUC
M

Transfection of
APCs Typical RNA Construct (for RNA vaccine)
5' UTR 3' UTR
CD4+
SA

Transfection of 5' cap CDS Poly A

keratinocytes CD8+
or Myocytes
iDC Lymph Node
Tc
Th
Immunity

Antigen
CD4+ B-cell

Apoptotic © Drawing Pin Publishing.

Bodies iDC

Fig B5. DNA and RNA vaccines design and principle of action.

28
B2. Plant Tissue Culture

into MS-Agar media and under the influence of plant hormones, undergoes callus development. Callus is a mass of
undifferentiated cells, which is formed by de-differentiation of explant cells. The process of de-differentiation is
complex and requires a well-tune, molecular events. Once the callus is formed, it is cut into smaller pieces and
shifted to new culture vessels with fresh culture media, containing a relatively high proportion of Auxin: cytokinin. In
this ratio plant hormones promote the differentiation of callus into roots and the process is called rooting. Now,
these rooted callii are transferred again into a fresh media containing a high proportion of cytokinin: auxin hor-
mones, which promotes shoot development, in a process called shooting. The mini-plantlets are now ready, yet
they need to be prepared for the release into the field, for which they are grown in a controlled environment in
sterile soil under green house for acclimatization. This process is known as hardening. Following hardening the
plants are ready for being released into the fields. The process of micropropagation helps in generating plants of
those plants which have no other means of propagation, or they do not produce viable seeds or they produce seeds

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at long interval (several years).

Surface
sterilization Place in MS-
(0.1% HgCl2) Agar media

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Explant

Callus Undifferentiated
Mass of cells
PA Hormonal Control

Callus
Shoots

Cytokinin
Roots
E
Friable Shooty Embryonic

Split Single callus

into multiple pieces Auxin
PL

Solid Rooty

Release
into fields
Place each part into
separate vessel
M

Hardening
Rooting Shooting in Greenhouse
SA

Micropropagated
© 2022. Drawing Pin Publishing.
calli

Fig B12. Outline of micropropagation process.

4.1.1 Role of plant hormones

The plant growth regulators are plant hormones involved in the plant’s growth and development. The five main
classes of plant hormones include auxin, cytokinin, gibberellin, ethylene, and abscisic acid. These growth regulators
are directly or indirectly involved in the regulation of growth and organized organ development of plant tissues
cultured under in vitro or laboratory conditions. The plant hormones auxin and cytokinin are particularly critical for
plant regeneration in tissue culture, with cytokinin playing an instrumental role in shoot organogenesis while
auxin playing role in root development and embryogenesis. Both are required for cell division and play roles in

43
 CHAPTER

III- Animal Cell

Culture B

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1. Introduction
Animal Cell, tissue and organ can be cultured like plant cell, tissue and organs on artificial media in controlled

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conditions. A cell differentiates and grows into a large number of cells. Although it should be known that while
all the plant cells are totipotent, animal cells are not and hence generating a complete organism of any type of
tissue or organ from any cell is not easy in animal cell culture in contrast to plant tissue culture. Now-a-days
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several valuable products of medical use have been produced through animal cell culture or genetically engi-
neered cells. The techniques have been much improved with the development of culture media, creation of
microbe-free environment and controlled artificial conditions. The history of animal cell culture might be
considered to begin with the Claude Bernard’s proposal in 1878 that physiological systems of an organism can
be maintained in a living system after the death of an organism. Later, in 1885, Roux maintained embryonic
E
chick cells in a saline culture and in the following year, Loeb demonstrated the survival of cells isolated from
blood and connective tissue in serum and plasma. However the most significant discovery towards opening
doors of animal cell culture was made in 1907, when Ross Harrison made the first attempt to culture animal
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cells wherein he cultivated embryonic nerve cells of frog by using hanging drop method. After supplementing
with chick embryo plasma, cells proliferated better. He is therefore known as father of animal cell culture. In
1910, Burrows succeeded in long-term cultivation of chicken embryo cell in plasma clots. He made detailed
ob-servation of mitosis, while in the next year, Lewis and Lewis made the first liquid media consisted of sea
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water, serum, embryo extract, salts and pep-tones. They observed limited monolayer growth. In the year
1913, Carrel introduced strict aseptic techniques so that cells could be cultured for long periods. In 1916, Rous
and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells. In another important
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discovery, Carrel and Baker developed ‘Carrel’ or T-flask as the first specifically designed cell culture vessel in
1923. In 1927, Carrel and Rivera produced the first viral vaccine – Vaccinia. In 1933, Gey developed the roller
tube technique. In 1940s, the use of the antibiotics penicillin and streptomycin in culture medium decreased
the problem of contami-nation in cell culture. Animal cell culture gained eminence after the use of viruses for
producing vaccines in late 1940s, and became indispensable after the production of polio vaccine in cultured
cells. Two factors made the cell culture development possible and led to the growth of this domain, first, the
development of antibiotics that made it easier to avoid many of the contamination problems that plagued
earlier cell culture attempts. Second was the development of the techniques, such as the use of trypsin to
remove cells from culture vessels, necessary to obtain continuously growing cell lines (such as HeLa cells).
Third, using these cell lines, scientists were able to develop standardized, chemically defined culture media
that made it far easier to grow cells. Later in 1948, Fischer developed a chemi-cally defined medium, CMRL
1066.

In 1952, Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane)
B3. Animal Cell Culture

1 ml of
Confluent cell Trypsin EDTA Add Media Transfer suspension
culture vessel to inactivate into 15 ml tube
trypsin Spin at 200g 15
for 5 min ml

Decant off Incubate untill

media cells detach
Wash
with PBS

ES
Perform viability
check 15
ml
Incubate for growth Plate the cells into
culture vessel
© 2022. Drawing Pin Publishing.
at specific density

G
Fig B20. Procedure of subculture
PA
Spin at 200g for
3-5 min to Wash the cells
pellet the cells with PBS
E
15 15
ml ml
15 Perform viability
ml
Transfer vial content check
PL

into 15 ml conical
Place the cryovial tube with pre-warmed
at 37oC waterbath culture media
Plate the cells into
culture vessel
Trypsinize and
at specific density
M

subculture
Original Cells
retrieved from stock
Incubation
SA

Trypsinize
and collect in
tube

Cool sequentially
-20oC, -80oC and 15
finally store ml

in gas phase Wash pellet

of liquid N2 and add © 2022. Drawing Pin Publishing.
cryo-media

Fig B21. Procedure of cryopreservation and retrieval

61
 CHAPTER

I- Genetically
Modified Plants C

ES
1. Introduction
Organisms developed by integrating a gene or component from other species or phylogentically unrelated

G
organism is known as transgenic organism. The production of a transgenic organism involves altering the
genome so that a permanent change is affected and a new variant capable of carrying the transgene for
subsequent generations is formed. This is different from somatic cell gene therapy, in which the effects of the
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transgene are restricted to the individual who receives the treatment. On one hand this is performed for
several socio-economic benefits such as increased productivity or pharmaceutical importance, on the other
side, however, this is an area of genetic engineering that has caused great public concern, and there are many
complex issues surrounding the development and use of transgenic organisms. In addition, the scientific and
technical problems associated with genetic engineering in higher organisms are often difficult to overcome.
E
This is partly due to the size and complexity of the genome, and partly due to the fact that the development of
plants and animals is an extremely complex process that is still not yet fully understood at the molecular level.
Despite these difficulties, methods for the generation of transgenic plants and animals are now well estab-
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lished, and the technology has already had a major impact in a range of different disciplines. Trangenesis is
the phenomenon of creating whole point of generating a transgenic organism is to alter the germ line so that
the genetic change is inherited in a stable pattern following reproduction. Trangenesis has been performed
on inter-phylum as well as inter-kingdom level. Another similar approach is called cisgenesis (also called
M

intragenesis), in which the genes are transferred either within the host species or sexually compatible spe-
cies. The new genes are introduced using recombinant DNA methods and gene transfer.
SA

Herbert Boyer and Stanley Cohen created the first genetically modified organism in 1973. They isolated a gene
from a bacterium that provided resistance to the antibiotic kanamycin, inserted it into a plasmid and then
induced another bacteria to incorporate the plasmid. The bacteria was then able to survive in the presence of
kanamycin. Boyer and Cohen expressed other genes in bacteria. This included genes from the toad Xenopus
laevis in 1974, creating the first GMO expressing a gene from an organism from different kingdom. First
genetically modified plant was produced in 1983 by Michael W. Bevan, Richard B. Flavell and Mary-Dell Chilton.
They infected tobacco with Agrobacterium transformed with an antibiotic resistance gene and through tissue
culture techniques were able to grow a new plant containing the resistance gene. The first field trials of
genetically engineered plants occurred in France and the USA in 1986, when tobacco plants were engineered
to be resistant to herbicides. In 1987, Plant Genetic Systems (Ghent, Belgium), founded by Marc Van Montagu
and Jeff Schell, was the first company to develop genetically engineered (tobacco) plants with insect tolerance
by expressing genes encoding for insecticidal proteins from Bacillus thuringiensis (Bt). The People’s Republic
of China was the first country to allow commercialized transgenic plants, introducing a virus-resistant tobacco
in 1992. The first genetically modified crop approved for sale in the U.S., in 1994, was the FlavrSavr tomato,
 Module 12: Applied Biology

which had a longer shelf life. In 1994, the European Union approved tobacco engineered to be resistant to the
herbicide bromoxynil, making it the first commercially genetically engineered crop marketed in Europe. In 1995, Bt
Potato was approved safe by the Environmental Protection Agency, making it the first pesticide producing crop to be
approved in the USA. Since the creation of first genetically modified organism, more than a dozen animals and
plants have been genetically engineered and successfully commercialised. We will be discuss more about some of
the popular genetically modified plants and animals in following sections.

2. Strategies for Developing Transgenic Plants

For thousands of years humans have manipulated the genetic characteristics of plants by selective breeding. This
approach has been extremely successful and will continue to play a major part in agriculture. However, classical
plant breeding programmes rely on being able to carry out genetic crosses between individual plants. Such plants

ES
must be sexually compatible (which usually means that they have to be closely related); thus, it has not been
possible to combine genetic traits from widely differing species. Availability of genome sequences and several
hybridization and DNA amplification techniques have led to the development of improved breeding methods that rely
upon DNA based markers such as SSR, RAPD, RFLP and AFLP etc. (Discussed in more detail in chapter 12F).

G
Agrobacterium Method Agrobacterium Method
(Preferred for dicots) (Preferred for monocots and dicots)
Agrobacterium
tumefaciens
Ti-Plasmid
carrying
a desired gene
PA Particles coated
with DNA encoding
desired gene

Gene
gun
Co-cultivation of
trangenic Agrobacterium Bombardment of
E
with plant cells plant pieces
with DNA containing
particles
PL

Foreign gene
M

Transformed plant cell

Callus Induction
SA

Organogenesis Development of plant

© 2018. Drawing Pin Publishing

Fig C1. General strategy for development of transgenic plants.

The drawback of conventional breeding programs is that they are time consuming and less probable for a desired
trait. This problem has been resolved to a great extent by advent of genetic engineering. With existing tools of
genetic engineering, agricultural scientist have a very powerful way of incorporating defined genetic changes into
plants and improve the traits in targeted manner, which is less time consuming and less labour intensive. Most of
the changes are aimed at improving the productivity and ‘efficiency’ of crop plants, both of which are important to
help feed and clothe the increasing world population. In some cases ornamental value or aesthetic sense have also
been improved using genetic engineering tools. There are two main requirements for the successful genetic ma-

72
 CHAPTER

II- Genetically
Modified Animals C

ES
1. Introduction
Trangenesis or introduction of foreign gene into an animal and its stable maintenance to cause a heritable

G
pattern leads to formation of transgenic animals. Several genetically engineered animals have been created
so far by researchers, unlike plants, most of them remain for the use in research. A very few have been
commercialised and approved for human use and release in environment. A large sect of transgenic animals
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include animal models for diseases used in research. Transgenic animals are routinely used in the laboratory
as models in biomedical research. Over 95 per cent of those used are genetically modified rodents, predomi-
nantly mice. They are important tools for researching human disease, being used to understand gene function
in the context of disease susceptibility, progression and to determine responses to a therapeutic intervention.
Mice have also been genetically modified to naturally produce human antibodies for use as therapeutics.
E
Seven out of the eleven monoclonal antibody drugs approved by the FDA between 2006 and 2011 were
derived from transgenic mice. The ability to produce transgenic animals is reliant on a number of components.
One of the first things needed to generate transgenic animals is the ability to transfer embryos. The first
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successful transfer of embryos was achieved by Walter Heape in Angora rabbits in 1891. Another important
component is the ability to manipulate the embryo. In vitro manipulation of embryos in mice was first reported
in the 1940s using a culture system. What is also vital is the ability to manipulate eggs. This was made
possible through the efforts of Ralph Brinster, attached to the University of Pennsylvania, who in 1963 devised
M

a reliable system to culture eggs, and that of Teh Ping Lin, based at the California School of Medicine, who in
1966 outlined a technique to micro-inject fertilised mouse eggs which enabled the accurate insertion of
foreign DNA.
SA

The first genetic modification of animals was reported in 1974 by the virologist Rudolph Jaenisch, then at the
Salk Institute, and the mouse embryologist Beatrice Mintz at Fox Chase Cancer Center. They demonstrated the
feasibility of modifying genes in mice by injecting the SV40 virus into early-stage mouse embryos. The result-
ing mice carried the modified gene in all their tissues. In 1976, Jaenisch reported that the Moloney Murine
Leukemia Virus could also be passed on to offspring by infecting an embryo. Four years later, in 1980, Jon
Gordon and George Scango together with Frank Ruddle, announced the birth of a mouse born with genetic
material they had inserted into newly fertilised mouse eggs. By 1981 other scientists had reported the suc-
cessful implantation of foreign DNA into mice, thereby altering the genetic makeup of the animals. This
included Mintz with Tim Stewart and Erwin Wagner at the Fox Chase Cancer Center in Philadelphia; Brinster
and Richard Palmiter at the University of Washington, Seattle; and Frank Costantini and Elizabeth Lacy at
Oxford University.

Most of the transgenic models were initially developed using mouse, hence most of our discussion about
 Module 12: Applied Biology

purpose by delete the gene responsible for the human rapid immune rejection response. In Canada, a National
survey on xenotransplantation showed that only 48% found acceptable for ‘the use of animals as a source of living
cells, tissues or organs to prolong human life. To overcome the Hyperacute rejection & acute vascular rejection,
synthesis of human regulators of complement activity are produced in transgenic pigs. Survival rates, after the
transplantation of porcine hearts or kidneys expressing transgenic regulators of complement activity proteins to
immunosuppressed nonhuman primates, reached near about 23 to 135 days. So, the Hyperacute rejection can be
overcome in a clinically acceptable manner. For long term graft tolerance induction of permanent chimerism via
intraportal injection of embryonic stem (ES) cells or the co-transplantation of vascularised thymic tissue. Transgenic
swine are used to produce human haemoglobin. The protein obtained from transgenesis could be purified by using
procine blood which is similar to human haemoglobin.

4. Examples of Transgenic Animals

ES
World’s first transgenic animal was created in 1974 by Rudolf Janisch by successful insertion of SV40 viral DNA into
mouse genome, however the rDNA did not express in the host. Later in 1982 first transgenic mice with expressing
rDNA was developed and was called supermice. It expressed mice growth hormone under human promoter transgene.

G
Till date a large number of transgenic animals have been created to fulfil the aforementioned purposes. Although a
larger set of transgenic animal still remains as mice and other models of diseases for scientific experiments, a few
have been created for commercial advantages such as improved food, ornamental value or conservation. Some
examples of popular successful transgenic animals are provided below.

Oncomice
PA First transgenic animal
Glofish
First Transgenic animal to be commercialised.
Created by placing Developed by expressing First Transgenic
an oncogene under fluorescent proteins animal
MMTV promoter
approaved for
E
(expressed in mammary
Dr. Zhiyuan Gong human consumption.
glands)
Leder and Stewart NUS, 1999 Created by
Harvard, 1984 insetion of GH
PL

gene of chinook
salmon
Supermice

Palmiter et al, 1984 Aqua-advantage salmon

Transgenic
Animals Aqua Bounty, USA, 2015
M

Created by placing a Chan etal,

Gen Pharm
growth hormone under Oregon Uni.,
International
human metallothionin (MMT) 2000
1991 ANDi
SA

promoter and thus had

large size. Developed by
microinjecting GFP gene
using viral vector
Developed by introducing
into a monkey.
human lactofrerrin
gene into bull for
© 2022. Drawing Pin Publishing Herman its constitutive expression

Fig C14. Examples of popular transgenic animals.

4.1 Oncomouse
The OncoMouse or Harvard mouse is a type of laboratory mouse (Mus musculus) that has been genetically modified
to have an active cancer gene and hence as model for studying cancer. In early 1983 Harvard University scientists
Philip Leder and Timothy Stewart created OncoMice by using a fine glass needle to inject known cancer genes into
mouse embryos just after fertilization. Oncomice carries a specific gene called an activated oncogene v-Ha-ras
under the control of the mouse mammary tumor virus promoter. The activated oncogene significantly increases the

90
 CHAPTER

III- Molecular
Diagnostics C

ES
1. Introduction
Molecular diagnostics refers to laboratory tests used to identify a disease, or the predisposition to a disease,

G
by analysing DNA- (deoxyribonucleic acid) or RNA (ribonucleic acid) or their proteins, in humans or, in the case
of infections, in microbes. Molecular diagnostic technologies have are known to play an important role in the
practice of medicine, public health, pharmaceutical industry, forensics and biological warfare in the 21st cen-
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tury. Some of these technologies include nucleic acid amplification like the polymerase chain reaction (PCR),
fluorescent in situ hybridization (FISH), peptide nucleic acids (PNA), and electrochemical detection of DNA,
biochips, nanotechnology and proteomic technologies. Many applications of molecular diagnostics are for
infections but are now increasing in the areas of genetic disorders, pre-implantation screening and cancer.
The birth of molecular diagnostics has been a little ambiguous and for a long time, some people have dated it
E
to the elucidation of the double helical structure of DNA by Watson and Crick (Watson and Crick, 1953) as that
revolutionary discovery provided scientists with the molecular tool that paved the way for molecular biology
research. However, it took scientists many more years to use that knowledge to have a significant impact on
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clinical medicine. One of the earliest FDA (US Food and Drug Administration) approved molecular diagnostics
tests was in 1985, which was a nucleic acid-based test for Legionnaire’s disease. Following that, most of the
other tests that were licensed in the 1980s and 1990s were targeted mainly at infectious diseases, namely,
HIV (human immunodeficiency virus), HPV (human papilloma virus), hepatitis B and C. In the area of infectious
M

diseases, diagnosis traditionally involved trying to detect and identify a microbe that is infecting the patient
using classical microbiological techniques such as viral and bacterial culture, and the use of antibodies.
SA

1 bn USD Use of NGS in Large-scale

Karry Mullis Roche aquired First PCR based
revenue from diagnostics covid -testing
invented PCR PCR from Cetus test for HIV
molecular diagnostics approved using RT-PCR
1985 1991 1998 2007 2014 2022

Development of Advent of Use of mass

real time PCR Next generation spectrometry
sequencing in diagnosis

Fig C22. Timeline of Molecular diagnostics (Redrawn after, Health Advances, Kristin Pothier, 2011)
 Module 12: Applied Biology

Chorionic Villi Sampling Amniocentesis

Transcervical Method Transabdominal Method

Ultrasound probe Ultrasound probe Ultrasound probe

Amniotic Fluid

Chorionic Chorionic

ES
villi villi

Ultrasound probe

G
Fluid (Supernatant) Biochemical
& Enzymatic studies
Centrifuge

Cells (Pellet)
PA Desquamated
cells
Molecular geneics
and cytogenetics

Fig C25. Methods of chorionic villi amniotic fluid samples commonly used for pre-natal molecular diagnosis.

----------------------------------------------------------------------------------------------------------------------------------
E
Brain Teaser

Q. which of the following is the least invasive method or least risky for the developing embryo?
PL

Aminocentosis, chorionic villi sampling, Sonography.

Hint: Sonography or ultrasound is least invasive

----------------------------------------------------------------------------------------------------------------------------------
M

3.3.1 Karyotyping –banding based

Karyotyping is the process of pairing and ordering all the chromosomes of an organism, thus providing a genome-
SA

wide snapshot of an individual’s chromosomes. Karyotypes are prepared using standardized staining procedures
that reveal characteristic structural features for each chromosome. A variety of tissue types can be used as a
source of these cells. For cancer diagnoses, typical specimens include tumour biopsies or bone marrow samples.
For other diagnoses, karyotypes are often generated from peripheral blood specimens or a skin biopsy. For prenatal
diagnosis, amniotic fluid or chorionic villus specimens are used as the source of cells. Without any treatment,
structural details of chromosomes are difficult to detect under a light microscope. Thus, to make analysis more
effective and efficient, cytologists have developed stains that bind with DNA and generate characteristic banding
patterns for different chromosomes. In 1970, when Torbjorn Caspersson and his colleagues described the first
banding technique, known as Q-banding. Q-banding involves use of the fluorescent dye quinacrine, which alkylates
DNA and is subject to quenching over time. This method is most useful for examining chromosomal translocations,
especially ones involving the Y chromosome.

Another approach to visualise chromosomes using dye is G-banding approach. In G-banding, metaphase chromo-
somes are first treated briefly with trypsin, an enzyme that degrades proteins, before the chromosomes are stained

110
 Module 12: Applied Biology

improperly; introducing a new gene into the body to help fight a disease. The commonest among these is replace-
ment gene therapy in which a single good copy of the gene is introduces to cure the defect. Furthermore, it would
obviously simplify treatment if the disease mostly affects just one or a few organs. The main steps involved in
replacement gene therapy are, (a) Identification and characterization of gene; (b) Cloning of gene; (c) Choice of
vector; (d) Delivery of gene into affected cell, and finally (e) expression of gene. The most important step involves
choosing a vector together with a suitable method of delivery. In addition, the vector/gene construct must be
designed to allow proper expression of the gene, once inside the patient. Delivery may be performed in a variety of
ways. The vector/gene construct may be injected into the bloodstream or other tissue. It may be aerosolized and
sprayed into the nose and airways (in vivo approach). In some cases, cells are removed from the patient, engi-
neered while growing in culture, and then returned to the patient (ex vivo approach) In general, a gene cannot be
directly inserted into a person’s cell. It must be delivered to the cell using a carrier, or vector. Two kind of vector

ES
systems often used in gene therapy are either, viral vectors or non-viral vectors. The overall process of two ap-
proaches of gene therapy is illustrated in the figure D14.

G
Gene Therapy

In vivo Approach In vitro Approach

Identify the gene of
interest for therapy PA Identify the gene of
interest for therapy

Clone and transfect

into a vector
Clone and transfect
into a vector
E

Isolate cells
PL

containing Retroviral Vectors

defective genes

Screen stem
Adenoviral Vectors
cells using
FACS
M

Transfect cells
Inject into the
SA

target Organ

Re-inject Culture on suitable

into target media
organ

Screen cells for

desired functional
gene copy
© 2022. Drawing Pin Publishing.
Grow in sufficient volume

Fig D14. Two main strategies for somatic cell gene therapy. For in vivo techniques the target gene is delivered directly to the
desired cell type within the patient. For ex vivo techniques, cells are removed from the body for delivery of the target gene, and then
re-implanted into the patient.

134
 CHAPTER

Biodiversity &
Bioresources E

ES
1. Introduction
Earth is a lively planet that not only supports life but also provides a huge number of resources for its suste-

G
nance. The complete life bearing component of Earth is known as biosphere. The biosphere is inclusive of life
supporting parts of atmosphere, hydrosphere and lithosphere. In applied biology, it is important to have
knowledge about the available resources and their distribution in order to maximize the benefits and keep a
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track of their status so that suitable conservation strategies may be applied. Although much of the basic
information on biodiversity originate from ecology (and hence described in standard ecology book), herein, we
will focus on the part that is essential for learning applied biology. In general, resources which are available
around us and help in maintenance of life are classified as perpetual, renewable and non-renewable. The
perpetual resources are those which will not be exhausted upto several millions of years, such as sunlight,
E
wind, flowing water etc. Renewable resources on the other hand are those which do not exhaust but their
usable form is generated by recycling and hence rate of consumption and recycling decide their direct avail-
ability.
PL

Planet Earth
M
SA

Resources Biodiversity

Varies Across the Globe

Perpetual Non-renewable
Limited reserviour Latitudnal Altitudnal
Unlimited shall exhaust in future Variation Variation
never be exhausted Renewable
Fossil Fuels
in near future Can be renewed Longitudnal
Wind, Sun and hence unlimited Variation
(balance needed)
Bioresources © 2022. Drawing Pin Publishing

Fig E1. Outline of bioresources and Biodiversity

Most of the biological resources lie in this category. Non-renewable resources on the other hand are vulner-
able to exhaustion, and they will not persist for ever, e.g. fossil fuels, minerals etc. It is important to learn
about available resources and their diversity to use them in the benefit of mankind and environment as a part
 Module 12: Applied Biology

2.2 Indian Biodiversity

With 2.4 per cent of the world’s land, India contributes 8 per cent to the world diversity. It has, therefore, been
designated as one of the 12 mega diversity regions and one among the 194 signatories to the Convention on
Biological Diversity (CBD) at Earth Summit in Rio de Janeiro in 1992. As per the international ‘biome’ type of
classification based upon climate, fauna and flora and the soil conditions, India can be divided into ten different
biogeographic zones as shown in the figure xx. India displays significant biodiversity. One of seventeen megadiverse
countries, it is home to 7.6% of all mammalian, 12.6% of all avian, 6.2% of all reptilian, 4.4% of all amphibian,
11.7% of all fish, and 6.0% of all flowering plant species. Readers are advised to visit https://indiabiodiversity.org/
for a comprehensive dataset of around 58.3 K species of India.

2.3 Latitudinal Gradient of Biodiversity (LGB)

ES
Latitudes are the imaginary horizontal lines across the circumference of the earth, with 0o latitude at the middle of
the sphere called equator and others with increasing values towards poles. The poles are located at 90o latitudes.
In classical assay of Malthus, he described in 1876 that “animal life is, on the whole, far more abundant and more
varied within the tropics than in any other part of the globe, and a great number of peculiar groups are found there

G
which never extend into temperate regions”. This evidence of latitudinal variation has been verified by several
scientific observations and it is now known that about 90% of the biodiversity is located in the tropical regions. Thus,
a latitudinal gradient means, a gradient involves high species’ numbers near the equator (at low latitudes) and
PA
lower numbers of species at high latitudes. Several environmental parameters form latitudinal gradients reflecting
the shape of the planet, its rotation and orientation to the sun (Fig
E
PL
M

Gradient of Biodiversity
SA

Tropics as cradle
Tropics as Museum Out of Tropics
Equator Equator Equator
Origination/Extinction Rate

Origination/Extinction Rate
Origination/Extinction Rate

90 S
o
Latitude 90o N 90 S
o
Latitude 90o N 90 S
o
Latitude 90o N

Fig E4. Latitudinal Gradient of Biodiversity

146
 CHAPTER

Animal and Plant

Breeding F

ES
1. Introduction
Animal and plant breeding has been done by humans since ancient times, perhaps approximately 10000 years

G
ago, since humans started agriculture. However, the ever-increasing population, created ennormous de-
mands and lead to more vigorous efforts on plant and animal breeding. It is projected that global populations
by 2050 will increase by one-third and as such will require a 70% increase in the production of food, hence
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improving the production of food is the need of an hour. In conventional terms, the breeding is to produce
animals and plants and select them for better traits. In case of plants, better traits may be high productivity,
insect resistance, pest resistance, herbicide tolerant etc., while in case of animals the trait improvement may
mean enhancement of milk production, fibre production, meat quality etc. Breeding is not merely applicable to
domesticated animals and agricultural plants but also proved to be very useful and essential in scientific
E
research.
PL

Breeding strategies for Plants and Animals

M

Conventional Apporaches Intermediate Approaches Modern Approaches

SA

Selection Breeding Polyploidy Protoplast Marker Genetic

FusionP Assisted Engineering
A
Selection Genome
MOET (MS) Editing
Mutagenesis

Outbreeding Interspecific
hybridization
Controlled
© 2022. Drawing Pin Publishing.
Breeding

Fig F1. Outline of methods used for conventional breeding.

One of the most commonly used strain of rat Rattus norvegicus (Spargue dawley) was an outcome of ennormous
breeding efforts, and today we have large repositories of experimental animals that have been bred and
genetically manipulated to generate disease and experimental models. The approaches to improve breeds
can be broadly categorised into three groups, one which are conventional and historical approaches, which
include conventional out-breeding and crossing the animals with known traits. Second, intermediate breeding
F. Plant and Animal Breeding

3. Molecular Markers (DNA based)

The characteristic detectable trait or feature revealing phenotype at the bimolecular level are referred to as the
molecular markers. Although there can be wide variety of molecular markers such as biochemical markers that
include small metabolites, proteins, lipids etc. In recent years recombinant DNA technology and PCR technology
have helped in construction of genetic, cytogenetic and physical maps of genomes of plants and animals molecular
markers. A specific DNA sequence revealing variations at DNA level and associated with a phenotype are referred
to as DNA markers. Use of DNA markers is more widespread in improvement of genetic traits and breeding
strategies, mainly due to the robustness and conserved nature of DNA markers. DNA based molecular markers are
genes or intergenic regions or repeats which are often found to be associated with specific trait desired for selec-
tion. DNA based molecular markers are not only useful in plant and animal breeding but also have a number of
additional applications such as DNA fingerprinting, strain identification (discussed in chapter 12C), phylogenetic

ES
analysis etc. In our discussion here, we will focus more on use of DNA markers as tool for plant and animal breeding
especially as marker assisted selection. Let us first understand, the different regions of genomic DNA that can be
chosen as molecular markers.

3.1 What could be a DNA marker?

G
Primarily any variation in the DNA that represents a marked difference in base pairs over a significant length and

PA
associate itself with a specific trait is a potential marker. A large number of genetic polymorphism occurring at DNA
sequence level can be used as molecular marker for evaluation of phenotypic variability. However a molecular
market must possess the following desirable properties;

· It must be polymorphic so that diversity must be measured.

· It should be evenly distributed throughout the genome.
E
· It should be easily and fast detected.
· It must distinguish the homozygotes and heterozygotes (not necessarily – if not fitting into this criteria, marker will be called as dominant,
else co-dominant).
PL

Components of Genome

Unique DNA
M

Repeated DNA
Less variation within population Large variation within population
SA

Interspread Repeats Tandem Repeats

Repeats that are not continous

Repeats that are continous

Short Interspread Long Interspread

100-200 bp long repeats 6-10 kbp long repeats
Alu Repeats L1, RTE, R2

Microsatellite Minisatellite Macrosatellite

© 2022. Drawing Pin Publishing. 1-13 bp repeats 11-60 bp repeats ~1000 bp repeats
SSR or STR or SSLPs VNTRs Gene Copies

Fig F4. Outline classification of potential DNA markers in genome.

167
 Module 12: Applied Biology

In order to identify potential DNA based molecular markers let us revisit a typical genome composition and try to
figure out the potential unique regions in DNA of individuals in a population. A typical genome contains a lot of
unique DNA, which is associated with several genes or regulatory elements, some of them may be conserved. The
unique DNA does not imply that it is unique in an individual but unique in a given genome (i.e. not repeated), but a
large fraction of it may be conserved across a population and hence not much useful as marker. However some
polymorphisms in the coding DNA (unique DNA) also exits that are useful as marker such as single nucleotide
polymorphisms or SNPs. But, another component of genome called repeated DNA varies largely across individuals
in a population (both in plants and animals) and hence more likely to fit in the criteria of marker. Repeats can further
be of two types, interspersed repeats and tandem repeats. Interspersed repeats include discontinuous repeats
distributed at a large distance in genome. These may be short size (100-300 bp), such as Alu1 repeats or large size
(6000- 10,000 bp) such as LINE. Tandem repeats are present continuously over a stretch of DNA without any

ES
significant gap or intervening region. They may further be categorized into three types based on the size of an
individual repeat, microsatellite (1-13 bp) such as short sequence repeats (SSR), minisatellite (11-60 bp) such as
variable number tandem repeats (VNTRs) and marcosatelites (~1000 bp) such as gene copies (CNVs). Fig F1
illustrates the potential DNA markers in genome of organisms. Apart from the canonical genomics regions like
repeated and interspersed regions, some other components of the genome which have been well characterized can

G
also be used as potential markers. Few such examples include STS, EST and SNPs.An STS is a short segment of
DNA which occurs but once in the genome and whose location and base sequence are known. STSs are detectable
by the polymerase chain reaction (PCR), are helpful in localizing and orienting mapping and sequence data, and
PA
serve as landmarks in the physical map of the genome. EST (expressed sequence tag) A unique stretch of DNA
within a coding region of a gene that is useful for identifying full-length genes and serves as a landmark for
mapping. An EST is a sequence tagged site (STS) derived from cDNA.

Note: A tag may be considered analogous to tags that are present on our clothes and help us to recognize their
brands. As the tag is unique to a brand, that’s characteristic and fool proof method to recognize it. However, color
E
texture and fabric may be same for different brands, similarly other regions of DNA may also be same, but tags are
unique hence widely used.
PL

In order to qualify a genomic element as molecular marker it must be associated with a trait such as a normal plant
as well as a draught resistant plant both may have a number of repeats in their DNA, so an individual repeat may not
be called as a marker, but as their position and number may vary, therefore the set of repeats and their pattern
could be considered as a marker for draught resistance. Hence, it is not merely simple PCR of a gene that would be
M

helpful for using these markers to identify a trait in plant or animals but different strategies that can actually identify
the patterns would be useful in trait selection and therefore marker assisted selection. In the following section we
will discuss more about the strategies for identification of aforesaid molecular markers. Some of which also involve
SA

the use of polymerase chain reaction (refer module 13, chapter A for more details on PCR).

3.2 Strategies for identifying a DNA based molecular marker

Molecular markers possess unique genetic properties on the basis of techniques used for detection. They are
classified into the two major classes: hybridization-based markers and PCR-based markers. The molecular markers
used for this effort are: (i) restriction fragment length polymorphisms (RFLPs), (ii) random amplified polymorphic
DNAs (RAPDs), (iii) minisatellite or variable number of tandem repeats (VNTRs), and (iv) microsatellites or simple
sequence repeats (SSRs). Besides, the contigs (contiguous sets of overlapping clones) used in cosmids and YAC
(yeast artificial chromosome) vectors also helped in construction of physical maps of genome. Use of RFLPs is the
non-PCR-based approach and the others are PCR-based approaches.

168
F. Plant and Animal Breeding

Detection of Polymorphic DNA (Molecular Markers)

Hybridization based detection PCR based detection

Involves southern transfer and PCR amplification of polymorphic
detection using probes DNA and its detection on gel

(Co-dominant Markers)

RFLP for RFLP for Arbitary Primer based Sequence specific

ES
SNP and SSR VNTR (AP-PCR) primer based
detection detection (Dominant Markers)
(Co-dominant Markers)

SCoT
ISSR Start
RAPD AFLP EST Codon

G
Inter Simple
Expressed Targetted
Random Amplification Amplified Fragment Sequence
Sequence
of Polymorphic DNA Length Polymorphism Repeats STS Tags SCAR
Sequence Sequence
Tagged
© 2022. Drawing Pin Publishing.

PA Sites
Characterized
Amplified Region

Fig F5. Outline of various strategies used for detection of DNA based molecular markers.

3.3 Restriction Fragment Length Polymorphism

E
Restriction fragment length polymorphisms, the first molecular marker developed by Botstein et al. RFLP involves
the use of restriction enzymes that cut genomic DNA molecules at specific nucleotide sequences (restriction sites),
PL

thereby yielding variable size DNA fragments. Difference in location of repeats or difference in genomic sequence
leads to different types of fragments. RFLP is a co-dominant marker in which the probes are usually small (500 to
3000 bp), cloned DNA fragments (e.g. genomic or cDNA. The RFLP is frequently used in genome mapping and in
variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.), and marker assisted
M

selection of plants and animals. RFLP is also the basis of DNA fingerprinting, commonly used forensic method that
forms the basis of paternity test, criminal identification etc (Discussed in chapter 12B).
SA

Advantages: Robust and widespread applications, SNPsor INDELs can create or abolish restriction endonuclease (RE) recognition sites,
thus affecting quantities and length of DNA fragments resulting from RE digestion. Hence, it most suitable for detecting INDEL and
SNPs.

Limitation: Isolation of sufficient DNA for RFLP analysis is time consuming and labour intensive. However, PCR can be used to amplify
very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in
a shorter time. An alternative name for the technique is Cleaved Amplified Polymorphic Sequence (CAPS) assay.

3.3.1 How RFLP is performed?

In RFLP, first the total DNA is isolated and then digested using restriction enzymes. TaqI and MspI are the most
useful enzymes for the identification of RFLPs. Both enzymes recognize four basepair sites, TaqI recognizes TCGA
and MspI recognizes CCGG. If nucleotides were randomly distributed across the genome, TaqI and MspI sites would
be distributed at average distances of 270 bp and 514 bp, respectively. Identification of genomic DNA fragments is
done by Southern blotting, a procedure whereby DNA fragments, separated by electrophoresis, are transferred to
nitrocellulose or nylon filter. In this, filter immobilized DNA is allowed to hybridize to radioactively labeled probe

169
 Module 12: Applied Biology

DNA. The filter is placed against photographic film, where radioisotope disintegration from the probe results in
visible bands. An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested
DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic
to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used
as RFLP probes.

3.3.2 Designing RFLP probes

The probes of RFLP are designed on the basis of fact that which fragments differ on gel between two conditions or
samples (as they can be considered as marker). In order to detect DNA fragments in gel, probes are needed. The
first step in RFLP probe design is digestion of total DNA with a methylation-sensitive enzyme (for example, PstI),
thereby enriching the library for single- or low-copy expressed sequences (PstI clones are based on the suggestion

ES
that expressed genes are not methylated). The digested DNA is size-fractionated on a preparative agarose gel, and
fragments ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for example, pUC18).
Digests of the plasmids are screened to check for inserts. Southern blots of the inserts can be probed with total
sheared DNA to select clones that hybridize to single- and low-copy sequences. The probes are screened for RFLPs
using genomic DNA of different genotypes digested with restriction endonucleases. Typically, in species with mod-

G
erate to high polymorphism rates, two to four restriction endonucleases are used such as EcoRI.

General Procedure Polymorphism detection using RFLP

Isolation of DNA
from desired source
Normal site
(specific cleavage
occurs)
PA
GAATTC
GAATTC
Polymorphic site
(Cleavage does
not occurs)
GTATTC
GAATAC
Change in
a single base

DNA digestion using

E
methylation sensitive Allele 1
restriction endonuclease
Allele 2
1. Homozygous Normal 2. Heterozygous 3. Homozygous Polymorphic
PL

Separation of fragments
using gel-electrophoresis
M

Autoradiogram
Southern transfer and Probe region
Corresponding
hybridization of specific fragments
probes for a given region
SA

Samples and their

polymorphism differentiated
as per autoradiogram 1 2 3
SDS-PAGE
© 2022. Drawing Pin Publishing.

Fig F6. Outline procedure of RFLP

Polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish
restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers.
Generally a restriction site is highly prone to non-recognition by enzyme in case of any change in sequence. Hence,
in case of polymorphic DNA, if one sample has a restriction site eg. G/AATTC, it will be recognised and cleaved by
enzyme EcoRI, while in another sample if there is an SNP (polymorphism at one base), changing this site to GTATTC,
will render this site non-function and hence enzyme will not cleave at that position in second sample. Such events
result in differences in the size of fragments obtained across polymorphic samples, thereby enabling the identifica-

170
 CHAPTER

Bioremediation &
Phytoremediation G

ES
1. Introduction
Bioremediation is defined as the process whereby organic wastes are biologically degraded under controlled

G
condition to an innocuous state, or to levels below concentration limits established by regulatory authorities.
By definition, bioremediation is the use of living organisms, primarily microorganisms, to degrade the environ-
mental contaminants into less toxic forms. It uses naturally occurring bacteria and fungi or plants to degrade
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or detoxify substances hazardous to human health and/or the environment. The microorganisms may be
indigenous to a contaminated area or they may be isolated from elsewhere and brought to the contaminated
site contaminant compounds are transformed by living organisms through reactions that take place as a part
of their metabolic processes. Biodegradation of a compound is often a result of the actions of multiple organ-
isms.
E

Bioremediation
PL

In-Situ
En-Situ
Bioremediation
Bioremediation
M

Intrinsic Extrinsic
Bioremediation Bioremediation Slurry Phase Solid Phase
Bioremediation Bioremediation
SA

Bioreactors Aerated Lagoons

Bioventing Biostimulation Landfarming Biopiling

Biosparging Composting
Bioslurping
Bioaugmentation © 2022. Drawing Pin Publsihing

Fig G1. Outline Classification of various bioremediation strategies.

For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them
to harmless products. As bioremediation can be effective only where environmental conditions permit micro-
bial growth and activity, its application often involves the manipulation of environmental parameters to allow
 Module 12: Applied Biology

from the vadose zone, and bioventing to stimulate biodegradation of less volatile hydrocarbons in unsaturated and
capillary zones. By removing free product and addressing residual contamination in the same step, bioslurping can
increase efficiency and lower costs and treatment times when compared to phased hydrocarbon remediation
techniques.

Bioventing Biosparging
Vacuum Pump Vapour
Extraction Well
Injection Vapour Blower
Well Low Rate Treatment Injection
Monitoring Point Well
Air-Injection
Blower
Water Vadose

ES
Table Zone
Smear Zone

Saturated Zone

Contaminated Soil
Contaminated Water
Water Table

G
Bioslurping
Bioaugmentation
Hydrocarbon Air
Hydrocarbon water Treatment
separator Cell Bioaugmentation Gene Bioaugmentation
Water

Contaminated
Soil
Pump PA Mixing of microbial
inoculum in soil
Activated soil

Bacterial constructs
of genetic elements Naked
constructs

Water Table
Contaminated
Soil
E
Fig G4. Scheme of various engineered in situ bioremediation methods- a. bioventing, d. biosparging, and e. bio slurping and
bioaugmentation.
PL

3.2.4 Bioaugmentation

Bioaugmentation is the method of application of autochthonous or allochthonous wild type or genetically modified
microorganisms to contaminated area. Bioaugmentation is commonly used in municipal wastewater treatment to
M

restart activated sludge bioreactors. Most cultures available contain microbial cultures, already containing all neces-
sary microorganisms (B. licheniformis, B. thuringiensis, P. polymyxa, B. stearothermophilus, Penicillium sp., As-
pergillus sp., Flavobacterium, Arthrobacter, Pseudomonas, Streptomyces, Saccharomyces, etc.). Activated sludge
SA

systems are generally based on microorganisms like bacteria, protozoa, nematodes, rotifers, and fungi, which are
capable of degrading biodegradable organic matter. There are many positive outcomes from the use of
bioaugmentation, such as the improvement in efficiency and speed of the process of breaking down substances and
the reduction of toxic particles in an area.

3.2.4 Bio stimulation

Biostimulation is the addition of nutrients to the contaminated area which enhances the metabolic activity of the
indigenous microbial community. Biostimulation enhances the metabolic activity of the indigenous microbial commu-
nity through nutrients amendment. Nitrogen and phosphorus are the essential growth-limiting nutrients for micro-
organism growth. Many studies suggest that the supplement of nitrogen and phosphorus is important to enhance
the degradation of petroleum 45 in polluted soil. Studies have indicated that when the C/N/P ratio is regulated to
100/5/1, 100/10/1, and 100/15/1, the bacterial activity in bio stimulation practice is tolerant to various hydrocar-
bons and can utilize hydrocarbons as carbon sources for their growth. Since different properties of contaminated
soil has a diverse microbial community, the optimum C/N/P ratio may be different for remediation. Generally, a ratio

188
 Module 12: Applied Biology

------------------------------------------------------------------------------------------------------------------------------------------------------------------
Brain Teaser

Q. How to perform a biochemical analysis of biodegradation?

To provide experimental proof of biodegradation during composting, a common hazardous contaminant pesticide, 14C-labelled Carbaryl was added
in sewage sludge-wood chip mixture at 1.3 – 2.2 ppm concentration. After 18 – 20 days in laboratory composting apparatus, 1.6 – 4.9 percent of
Carbaryl was recovered as 14CO2 and remaining bound to soil organic matter.

----------------------------------------------------------------------------------------------------------------------------------

4.1.2 Land farming

Land farming is an ex-situ waste treatment process that is performed in the upper soil zone or in bio treatment cells.

ES
Contaminated soils, sediments, or sludges are transported to the land farming site, mixed into the soil surface and
periodically turned over (tilled) to aerate the mixture. Land farming commonly uses a clay or composite liner to
intercept leaching contaminants and prevent groundwater pollution, however, a liner is not a universal requirement.
This technique has been used for years in the management and disposal of drill cuttings, oily sludge and other
petroleum refinery wastes. The equipment employed in land farming is typical of that used in agricultural opera-

G
tions. These land farming activities cultivate and enhance microbial degradation of hazardous compounds. During
land farming, the waste materials are typically placed as a layer on the ground surface with variable thickness. The
waste is then tilled and amended with nutrients to enhance biodegradation by naturally occurring bacteria. Fertil-
PA
izers such as urea and triple superphosphate (TSP) are used to provide nitrogen and phosphate necessary for
biodegradation. Reduction in hydrocarbon concentrations can be expected within a span of weeks to months,
depending on the initial concentration and composition of hydrocarbons, and the soil conditions. Land farming is a
low-cost technology. Facilities are simple to construct and easy to operate. Standard construction and farming
equipment can be used to move soils to the land treatment facility. Some limitation of land farming are large space
E
requirements, uncontrolled conditions particularly for recalcitrant compounds inorganic contaminants and the pres-
ence of metal ions that may be toxic to microbes and may leach from the contaminated soil into the ground.
PL

Hydrocarbon compounds that have been identified as being not readily degraded by land farming include creosote,
pentachlorophenol (PCP), and bunker C oil.

Land Farming
Composting
M

Nutrients Water
Tilling

Sand Layer
SA

Soil
Pit
Other organic waste Gravel Drainage Pipes
Vegetable refuge
Sand
Biopiling Leachate Collection
Gravel Soil Vapour and Treatment
Monitoring Air inlet/ (Optional)
Contaminated soil System Exhaust
Air injection
or excretion
Grid based
classical compost
Berm

Nutrient & Moisture

addition
© 2022. Drawing Pin Publsihing

Fig G5. Schematic of various ex-situ approaches of bioremediation a. Biocompost, b. land farming, and c. biopiling

190
 CHAPTER

Biosensors and
their Applications H

ES
1. Introduction
The term biosensor is used to describe a biological sensing device made up of a transducer and a biological

G
element that may be an enzyme, an antibody, or a nucleic acid, wherein during the sensing process, the
biological element interacts with the analyte being tested and the response is converted into an electrical
signal by the transducer. A standard definition of biosensor provided by regulatory committee states that
PA
“biosensor is an analytic device that consists of a biologic component in intimate contact with a physical
transducer component that converts the signal generated by the biologic component into a measurable elec-
trical or optical signal” Also, A.turner, defined a biosensor as “a compact analytical device incorporating a
biological or biologically derived sensing element either integrated within or intimately associated with a
physicochemical transducer” . In order to call a senor as biosensor, it must have a biological component that
E
acts as the sensor and an electronic component that detects and transmits the signal. In other words, the
biological material is immobilized and a contact is made between the immobilized biological material and the
transducer. The analyte binds to the biological material to form a bound analyte, which in turn produces the
PL

electronic response that can be measured. Sometimes the analyte is converted to a product that could be
associated with the release of heat, gas (oxygen), electrons, or hydrogen ions. The transducer then converts
the product-linked changes into electrical signals, which can be amplified and measured. We will discuss more
about the operating principle and types of the biosensors in subsequent sections.
M

M.Cremmer Leland C. Clark S. Girbi et. al.

SA

noted the electric W.S Huges developed the first First immunobiosensor demonstrated
potential arising developed biosensor for oxygen was developed by nerve-on-chip type
between a pH measurement later named Roederer and Basti biosensor for assessment
parts of the fluid device Clark’s electrode using PZ crystals of nerve impulse conduction
1906 1922 1956 1983 2018

1909 1936 1975 1999

Søren Sørensen Griffin and First commercial Poncharal P et. al.
developed the concept Nelson biosensor was demonstrated
of pH and pH scale demonstrated developed by the first nano-biosensor
enzyme- yellow spring
immobilization instruments

Fig H1. Timeline of biosensors development.

H. Biosensors and their Applications

5. Classification and Examples of Biosensors

Biosensors can be classified into various groups, typically based on the nature of transducers used, or on the basis
of bio acceptor. Most common bio acceptors are enzymes and antibodies and hence two prominent categories are
popular on this criterion namely enzyme biosensors and immunosesnors. However, other bio acceptors such as
nucleic acids, proteins, carbohydrates are also being used giving rise to various classes of biosensors. The most
widely used classification scheme of biosensors still relies on the nature of transducers. Among the most common
categories of transducers are electrochemical, optical, calorimetric and piezoelectric. However, newer classes of
biosensors have also emerged such as magetoelectric biosensors, gravimetric biosensors etc. Fig H5 illustrates
some common types of transducers are described in following subsections with examples. Our main focus in this
discussion would remain on most popular biosensors, which include four main categories, viz, electrochemical
biosensors, optical biosensors, calorimetric biosensors and piezoelectric biosensors.

ES
Types of Bioesnors

G
On the basis of bioacceptors On the basis of detection system

Enzyme
biosensors Immuno-
PA Optical
biosensors Electrical
sensors nano
chromogenic sensors SPR
Nucleic acid biosensors Electronic biosensors
fluorogenic
biosensors luminogenic biosensors Thermal
whole-cell
biosensors
E
biosensors
PL

On the basis of technology and transducers

Electrochemical Electronic
biosensors biosensors Gravimetric
M

biosensors
Emerging
Thermal
Technologies
(calorimetric)
biosensors Lab on chip
SA

Optical Magnetic
Potentiometric Impedometric Nanosensors
biosensors
biosensors biosensors SPR
Voltametric Conductometric
sensors biosensors

Amperometric
biosensors

Fig H5. Outline classification of Biosensors on the basis of transducers (Adapted from Arya A et al).

5.1 Electrochemical Biosensors

The basis of electrochemical biosensors is to harness the electrical changes occurring during biochemical reactions,
which can be detected by either measurement of potential difference of direct current, further classifying these
biosensors as potentiometric or amperometric biosensors and Conductometric biosensors. A less popular variant of

209
H. Biosensors and their Applications

Amperometric Biosensor Cyclic Voltametry

Capacitive
current Epa
Faradic Anodic (oxidation)
(background)
Platinum Anode current -positive current
(analyte)
Electrode body ipa

Current (A)
Kcl solution
Silver Cathode
ipc Potential (V)
Immobilized enzyme

Semipermeable Cathodic(reduction) Epc

ES
membrane - Negative current

Ion selective field effect transistor Potentiometric

Biosensor

Glass body

G
Encapsulation
Gate
Contact Leads
Electrode

Source Drain
SiO2
PA
Substrate
Counter
electrode
pH sensitive membrane

pH sensitive membrane

Fig H6. Various electrochemical Biosensors and typical cyclic voltammetry curve.
E
------------------------------------------------------------------------------------------------------------------------------------------------------------------
Brain Teaser
PL

Q. What is impedometric biosensors? How do they work?

Hint: An impedimetric biosensor is constructed by immobilizing biological recognition elements onto an electrode surface. It reports, through
measurement and/or monitoring, the targeted analyte through the output of an electrical impedance signal made proportional to analyte activity.
M

----------------------------------------------------------------------------------------------------------------------------------

Table H1. Comparision between potentiometric, amperometric and conductometric biosensors

SA

Type Advantage Disadvantage

Potentiometric ISE translation is relatively easy Require reference electrode
Easily miniaturised Limited linear range, pH sensitive

Amperometric Wide variety of biochemical redox mechanisms Require reference electrode

Easily miniaturised Multiple enzymes may be required
Good dynamic range Multiple membranes required
Relatively good sensitivity

Conductometric Easy to fabricate Non-selective unless in array

No-reference electrode needed
Low frequency source

211
 Module 12: Applied Biology

First generation Second generation Third generation

Reactant Product Reactant Product Reactant Product

Bioacceptor Bioacceptor
No mediator
Mediator Mediator required
O2 Bioacceptor
H2O2 (oxidised) (reduced)

ES
- - - - - - - - -
e e e e e e e e e

Fig H11. Various generations and principle of biochemical reactions in a typical glucose biosensor.

G
----------------------------------------------------------------------------------------------------------------------------------

Brain Teaser
PA
Q. Name some common interfering agents in strip based glucose bio sensing ?

Hint: acetaminophen, salicylic acid, tetracycline, dopamine, ephedrine, ibuprofen, L-DOPA, methy-DOPA, tolazamide, ascorbic acid, bilirubin,
cholesterol, creatinine, triglycerides, and uric acid. Ascorbic acid and low haematocrit values are most significant interfering agents in glucose
biosensors.
E
----------------------------------------------------------------------------------------------------------------------------------
PL

6.4 Advancements in Glucose Biosensing

Beyond the three generations of glucose biosensors, a rapid advancement has been witnessed in glucose biosensing
in past one decade and researchers are interested in developing newer classes of glucose biosensors, which
include continuous monitoring systems, point of care systems and non-invasive glucose sensing devices. As diabet-
M

ics need a routine measurements, recurring puncture of skin with needle is painful and hence, such systems which
are non-invasive or can perform a glucose monitoring in continuous mode shall be highly valuable. Continuous ex
vivo monitoring of blood glucose was proposed in 1974, while in vivo glucose monitoring was demonstrated in 1982.
SA

Two types of continuous glucose monitoring systems are currently in use - a continuous subcutaneous glucose
monitor and a continuous blood glucose monitor. However, due to surface contamination of the electrode by proteins
and coagulation factors and the risk of thromboembolism, most of the CGMSs do not measure blood glucose
directly. Therefore, subcutaneously implantable needle-type electrodes measuring glucose concentrations in inter-
stitial fluid have been developed, which reflect the blood glucose level. Continuous subcutaneous glucose monitor-
ing can also be achieved without direct contact between the interstitial fluid and transducer by using the microdialysis
technique. GlucoDay (Menarini, Florence, Italy) and SCGM (Roche, Mannheim, Germany) are based on a microdialysis
technique.

Non-invasive glucose analysis is another goal of glucose sensor technology and significant efforts have been made
to achieve this goal. Optical or transdermal approaches are the most common noninvasive glucose sensing meth-
ods. The optical glucose sensors use the physical properties of light in the interstitial fluid or the anterior chamber
of the eye. These approaches include polarimetry, Raman spectroscopy, infrared absorption spectroscopy, photo

218
 APPENDIX

Previous Years’
Questions (CSIR) 1

ES
Microbial Fermentation

Q1. Which cycle has been used in Heterolactic fermentation?

G
1. Entner Duodoroff Pathway 2. Phosphoketolase Pathway

3. Pentose Phosphate Pathway PA 4. Glycolate Pathway

Solution (Correct Answer – 2): As we discussed in chapter A, Heterolactic fermentation is observed in microbes which are missing
crucial glycolytic enzymes aldolase and triosephosphate isomerase. Due to this microbes can not perform entner duodoroff
pathway. Thus, as an alternate pathway most microbes performing heterolactic fermentation follow phosphoketolase pathway (Fig
A2 for details).
E
Q2. The word fermentation is used in biochemistry and microbial technology to denote different phenomenon.
If the former is called C and the latter is called T, which of the following statement is true?
PL

1. All T is C but C is not T

2. All C is T but T is not C

3. T is always an aerobic product of genetic engineering while C is not

M

4. C is always an aerobic product while T can be either aerobic or anaerobic

SA

Solution (Correct answer - 3). In biochemistry mostly the fermentation process is used to depict the biochemical fates of pyruvate
to regenerate NAD+, which mostly occurs but not limited to anaerobic conditions. However, the term fermentation in microbial
technology is used for a large variety of other reactions which could be aerobic and even not result conventional energy metabo-
lism, rather generation of several other metabolites. Hence, it is true that all fermentation process of biochemistry are part of
fermentation in microbial technology but not vice versa (All C is T but not C).

Q3. During the production of alcohol by fermentation using budding yeast, oxygen supply is kept limited. Why?

1. Alcohol is oxidized further in the presence of oxygen

2. Budding yeast lose mitochondria in the absence of oxygen

3. Budding yeast are obligate anaerobes and cannot tolerate oxygen

4. Budding yeast are facultative anaerobes

Solution: (Correct answer -1). Louis Pasteur, in 1857 demonstrated that on aerating yeasted broth causes yeast cell growth to
C2. Genetically Modified Animals

UT T1 T2 T3 T4 T5 T6
control

Which one of the following options represents potential single copy event?

ES
1. T1, T5 and T6 2. T2 and T3

3. T4 only 4. 2 only

Solution: (Correct answer -1 ). As previously discussed in transgenic plants chapter, a single copy event would result in appearance of
single band (ignore the common band of untransformed UT control). Hence such bands are apperant in plant T1, T5 and T6 alone, so they

G
represent a single copy event.

PA
Q5. A student wrote following statements regarding comparision of restriction fragment length polyumorphism
(RFLP), Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP)and simple
sequence repeats (SSRs) techniques used for generating molecular markers in plants :

A. All these techniques can be used for fingerprinting

B. Detection of allelic variations can be achieved only by RFLP and SSRs.

E
C. Use of radioisotopes is required for RFLP and RAPD only
PL

D. Polymerase chain reaction is required for all the techniques

Which one of the following combinations of above statements is correct?

1. A and B 2. B and C
M

3. C and D 4. D and A

Solution: (Correct answer – 1). It is true that all the methods are useful for DNA fingerprinting, DNA fingerprinting is used for identification
SA

of an organism in a population (just like our own fingerprints), as they are unique. Similarly allelic variants i.e. dominant homozygous,
recessive homozygous or heterozygous can be determined using RFLP or SSR (even AFLP). RAPD cannot be used to differentiate between
homozygous and heterozygous alleles. Statement C and D are however incorrect, as RAPD does not require a radiolabel (detection is done
using PCR products run on a gel), and PCR is not needed for all the above techniques (RFLP in strict sense and conventional format does
not require PCR).

Q6. The figure below represent s a profile of DNA markers in two parents (P1 and P2), progeny (F1) from a cross
between P1 and P2and that of gametes produced from F1. Eight different patterns (DH1- DH8) were observed in
case of gametes. The numbers below, DH1 to DH8 indicate the number of observed in each case.

27
 T
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Notes NET exam preparation. This book provides a complete and
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detailed topic wise description of various application of

biology prescribed in the syllabus. This book has been edited by
highly experienced researchers with enormous experience in the
Applied domain. described, which makes it an ultimate unmatched piece of
Biology work representing several years of experience gathered from some of
Dr. Aditya Arya | Dr. Amit Kumar finest labs across the globe. The organization of the chapters is strictly
as per the syllabus in point to point manner. Besides direct applica-
tions, emphasis has been given fundamental principles and basis of
applied biology, and a number of sample problems have been added
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present throughout the text to provide a better learning experience.

ES
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