 Definition of Biotechnology

 Principles of Biotechnology
 Tools Of Biotechnology
 Traditional and modern biotechnology
 Advancement of biotechnology
 Application of biotechnology
 Scope of Biotechnology
 Biotechnology in India
 Gene therapy
 Bio Fertilizers
 Gene Bank
 Bibliography
 Definition of Biotechnology
Biotechnology is a term which is a combination of two individual
terms: Biology and Technology. As the name suggests “It is the
assembly of technology in science of biology”.

Most simply it may be defined as:

“The regulated and controlled use of the biological agents

for the manufacture of useful products or forgenerating
beneficial services.”

These biological agents may be microorganisms, animals or plants or

their cellular components. However, it is not easy to define
biotechnology in a single sentence because of its wide and
multidisciplinary applications. Various definitions have been given
by different scientific organization’s. One of such standard definition
as given by the ‘European Federation of Biotechnology’ is as follows:

Biotechnology is the integrated use of biochemistry,

microbiology and engineering sciences in order to achieve
technological applications of the capabilities of
microorganisms, IP cells/tissues and parts thereof.

U.S. National Science Federation says that “Biotechnology is the

controlled use of biological agents such as microorganisms
or cellular components for beneficial use.”
According to IUPAC (International Union of Pure and Applied
Chemistry), biotechnology means “the application of
biochemistry, biology, microbiology and chemical
engineering to industrial processes and products and on
environment.”
 Principles of Biotechnology
The field of biotechnology has evolved tremendously over the years. It has
gone from the traditional view of using microbes to generating products for
human use, to the modern view of using genes for developing vaccines.
Therefore, the European Federation of Biotechnology (EFB) has come up
with a new definition that involves both the traditional and the modern view
of biotechnology. It states that –

‘Biotechnology is the integration of natural science and organisms, cells, parts

thereof, and molecular analogs for products and services’. The two core
techniques responsible for the birth of modern biotechnology are:

 Genetic Engineering: It involves techniques that change the chemistry of the

genetic material such that when they are introduced into the host, the host
phenotype changes.
 Maintenance of a contamination-free environment to enable the growth of
only the desired microbe in large quantities during the manufacture of
products like vaccines, antibiotics etc.

Genetic Engineering
Traditional hybridization methods in plant and animal breeding introduce
desirable as well as undesirable genes. The techniques of genetic engineering
such as recombinant DNA technology, gene cloning etc. overcome this
problem, allowing us to introduce only desired genes. Genetically modifying
an organism has the following basic steps:

 Identification of DNA containing desired genes.

 Introduction of desired DNA into the host organism.
 Maintenance of the introduced DNA in the host genome and transfer of the
DNA to its progeny.
Let us now learn about the tools of biotechnology.
 Tools of Biotechnology
Genetic engineering or recombinant DNA technology is possible only if we
have some important tools. The following are some important tools of
biotechnology.

1) Restriction Enzymes
Restriction enzymes, also known as ‘restriction endonucleases’ are molecular
scissors that can cut DNA at specific locations. These are part of a larger class
of enzymes called ‘Nucleases’. Nucleases are of two kinds:

 Exonucleases – Enzymes that remove nucleotides from the ends of DNA.

 Endonucleases – Enzymes that make cuts at specific positions in the DNA.
‘Hind II‘ – the first restriction endonuclease to be isolated, identifies a specific
six base pair sequence and always cuts the DNA at a particular point. This
specific sequence is the ‘Recognition sequence‘. Today, there are more than
900 restriction enzymes, isolated from about 230 bacterial strains. Each of
these enzymes recognizes different recognition sequences. There is a naming
convention to name restriction enzymes. Let’s learn this using EcoRI as an
example:

 Eco – indicates that this enzyme was isolated from Escherichia coli.

 R – refers to the name of the strain.
 I – is the Roman numeral that indicates the order of isolation of this
restriction enzyme from the strain of bacteria.

Mode of Action of Restriction Enzymes

Endonucleases perform their function in the following manner:

 Inspect the length of DNA for the specific recognition sequence.

 Bind the DNA at the recognition sequence.
 Cut the two strands of DNA at specific points in their sugar-phosphate
backbone.
Restriction enzymes recognize specific palindromic nucleotide sequences in
the DNA. What is a palindrome? In terms of words, a palindrome is a group
of letters that produce the same word when reading forward or backwards. An
example is ‘RADAR’. In terms of DNA, a palindrome is a sequence of base
pairs that reads the same on both DNA strands when reading in the same
orientation. For example –

5′ —— GAATTC —— 3′
3′ —— CTTAAG —— 5′

On reading both the strands shown above in the 5′ to 3′ direction, they give
the same sequence. This is true even when they are both read in the 3′ to 5′
direction.

Restriction enzymes cut the DNA strand a little away from the center of the
palindromic site, but between the same two bases on both strands. This gives
rise to single-stranded, overhanging stretches on each strand called ‘sticky
ends’. They are called ‘sticky’ because they can bind to their complementary
cut counterparts.

Using restriction enzymes we can generate recombinant DNA that contains

DNA from different sources. Cutting these DNA sources with the same
restriction enzyme gives fragments with the same kind of ‘sticky ends’, that
can then be joined using DNA ligases.

Mode of action of EcoRI [Source: Wikimedia Commons]

2) Cloning Vectors
Just the way a mosquito acts as a ‘vector’ to transfer the malarial parasite into
the human body, we need vectors to transfer the cut DNA into a host
organism. Vectors are one of the important tools of biotechnology.

Plasmids make good vectors because they can replicate in bacterial cells,
independent of the control of the chromosomal DNA. The vectors in use
currently are engineered such that they help in easy linking of foreign DNA
and allow selection of recombinants over non-recombinants. A vector needs
the following features to enable cloning:

(i) Origin Of Replication (ori)

This is the sequence from where replication begins. Linking a piece of DNA
to this sequence causes it to replicate in the host cell. This sequence also
controls the copy number of the linked DNA. Therefore, the target DNA
needs to be cloned into a vector whose ‘ori’ supports high copy number, in
order to recover large amounts of the DNA.

(ii) Selectable Marker

The vector also needs to have a selectable marker which allows the selection
of recombinants over non-recombinants. In terms of E. coli, some useful
selectable markers are genes that provide resistance to antibiotics like
ampicillin, kanamycin, chloramphenicol etc. Since the normal E. coli cells do
not carry these resistance genes, it becomes easy to select the recombinants.

(iii) Cloning Sites

In order to attach the foreign DNA to a vector, the vector should have a
recognition site for a specific restriction enzyme. Multiple recognition sites
will result in multiple DNA fragments, complicating the process of cloning. A
vector has more than one antibiotic resistance gene. The foreign DNA is
ligated into a restriction site in one of the antibiotic resistance genes.

For example, let’s say an E. coli cloning vector has genes for ampicillin and
tetracycline resistance. On ligating the foreign DNA into a recognition site
within the tetracycline resistance gene, the plasmid loses its tetracycline
resistance. But, it can still be selected for non-recombinants by plating on
ampicillin-containing medium.
Now, by transferring the ones that grow in the ampicillin medium to a
medium with tetracycline, we can dissect out recombinants from non-
recombinants. The recombinants will grow in ampicillin but not in
tetracycline medium; while non-recombinants will grow in both mediums.

Currently, alternative markers are available that can differentiate

recombinants from non-recombinants based on their ability to produce color.
This involves insertion of the foreign DNA in the DNA sequence of an
enzyme like β-galactosidase, which inactivates the enzyme. This is
‘insertional inactivation’. On reaction with a substrate, the recombinants do
not produce color whereas non-recombinants produce color.

Blue-white screening using B-galactosidase [Source: tankonyvtar]

(iv) Vectors to Clone Genes in Plants and Animals

Long before us, bacteria and viruses knew how to transfer genes into plants
and animals. For example, Agrobacterium tumifaciens, a pathogen on dicot
plants, transfers ‘T-DNA’ that transforms normal plants cells into tumours.
These tumours then produce chemicals that the pathogen requires. With better
understanding, we have now converted these pathogens into useful vectors to
deliver genes of interest to the plants or animals.

Now, the tumour-inducing (Ti) plasmid of Agrobacterium tumifaciens has

been modified into a cloning vector. This vector is no longer harmful but is
useful in delivering genes of interest to plants. Retroviruses transform normal
cells into cancerous cells in animals. These have also been modified so that
they are no longer harmful and can deliver genes to animals.
3) Competent Host
Host cells are bacterial cells which take up the recombinant DNA. Since DNA
is hydrophilic, it cannot pass through the cell membrane of bacteria easily.
Therefore, the bacterial cells have to be made ‘competent’ to take up the
DNA.

Some procedures that make the cells competent are treatments with a specific
concentration of divalent cation like calcium. This makes it easy for the DNA
to enter the cell wall through pores. Incubation of cells with the recombinant
DNA on the ice, followed by heat shock at 42°C and another incubation on
ice, enables the cells to take up the DNA.

There are several other methods to introduce foreign DNA into host cells.
The ‘microinjection’ method involves injecting the recombinant DNA
directly into the nucleus of an animal cell.
The ‘bolistics’ or ‘gene gun’ method bombards plant cells with high-velocity
microparticles of gold or tungsten coated with DNA. The last method uses
‘disarmed pathogen vectors’ (discussed above) to transfer the recombinant
DNA into the infected host cells.
 Traditional and Modern
Biotechnology
The art of biotechnology is very old. It is as old as human civilization.
It actually began when man started the domestication of useful plants
and animals and started utilizing microbes for making various
beverages (like wine, beer), curd, vinegar, etc.

Alcohol was probably the first product of ancient biotechnology. Such

practices which have been in vogue since long by our ancestors and
are being used even today are included in the traditional
biotechnology. Such practices are very common in day-to-day life and
are also used in normal kitchen technology, i.e., while preparing idli,
dhokla, cheese, curd, etc.

With the advancement of science and technology, advent of new

analytical instruments and recent progress in the field of
microbiology, molecular biology, etc. it has become possible for us to
discover or improve better strains of microbes for commercial
production. This all comprises the modern biotechnology. In simple
words we may also differentiate between traditional (old) and
modern (new) biotechnology.

It is as follows:
Old Biotechnology is the one which involves the exploitation and
utilization of natural capabilities of microbes or cellular components
for manufacture of useful products or for services.

New Biotechnology involves the use of recombinant DNA technology,

enzyme engineering, genetic engineering practices, etc., for
developing newer or improved capabilities of biological agents for
production of beneficial services or products.
 Advancement of
Biotechnology
This branch of biology is in use by mankind since very long.
Numerous important achievements and advancements have been
made by many eminent workers for this discipline.

A few of such important contributions by various workers

in the field of biotechnology are enlisted below:

 Biotechnology as aMultidisciplinary Activity:

Biotechnology is truly multidisciplinary (or interdisciplinary) in
nature and it several disciplines of basic sciences and engineering.
The science disciplines from which biotechnology draws heavily are
Microbiology, Chemistry, Biochemistry, Genetics, Molecular Biology,
Immunology, Tissue Culture and Physiology.

Recent advancements have led to a multidisciplinary’ applicability of

biotechnology. Various areas in which this discipline is very
frequently used on large scale are: agriculture, food and beverage
industry, environment, medicines, energy and fuels, enzyme
technology, waste utilization, biodiversity conservation, etc. (Fig. 1).
Biotechnology has great impact in areas like Environment,
Bioinformatics, Genomics Proteomics and Human Genome Project
(HGP).
Applications of Biotechnology
Biotechnology is such a branch of science which has advanced rapidly
and has emerged as a potential science for providing benefits in all
the fields of human welfare. It has a great impact in almost all the
domains of human life, may it be health, environment, foods or
agriculture. Recent advancements have led to a multidisciplinary
applicability of biotechnology.

Various areas in which this discipline is very frequently

used on a large scale are as follows:
1. Agriculture

2. Food and Beverage Industry

3. Environment

4. Health care and Medicines

5. Energy and Fuels.

6. Enzymes and Biochemical.

7. Other Industrial applications.

8. Forensic cases

9. Conservation of Nature
 Scope of Biotechnology
Biotechnological approaches are applied to accomplish goals for the
benefit of mankind. Scientists have achieved many such goals and a
few fields are also there in which they are trying for success.

Following are a few programmes being undertaken by the

biotechnologists:
(a) Development of effective antiviral vaccines.

(b) Bio-control of plant diseases

(c) Genetically improving the pharmaceutical microorganisms.

(d) Large scale production of bio pesticides and bio fertilizers.

(e) Production of Human Interferon’s.

(f) Upgrading the photosynthetic efficiency of plants.

(g) Production of secondary metabolites from plants on large scale.

(h) Improved production of vitamins.

(i) Developing efficient biofuels.

(j) Developing methods for curing cancer.

(k) Better gene therapy practices for human.

(l) Production of transgenic animals and plants with better qualities.

(m) Protection of threatened species.

 Biotechnology in India
Like other developing countries, biotechnology has become a major
thrust in India also for promotion and planning of various
biotechnological programmes in India, there is present a separate
department called Department of Biotechnology (DBT).

DBT was set up in 1986 under the Ministry of Science & Technology.

DBT funds some important centres for exploiting biotechnological

approaches and also for promotion of postgraduate education and
research in the field of biotechnology. Apart from DBT there are
some other agencies also which work under the Indian Government
for promotion of biotechnological approaches in various fields like
industry agriculture and environment.

A few important of them are:

i. DST—Department of Science and Technology, New Delhi

ii. CSIR—Council for Scientific and Industrial Research, New Delhi

iii. ICMR—Indian Council of Medical Research, New Delhi

iv. IARI—Indian Agricultural Research Institute, New Delhi

There are many other centres in India which function, in one way or
the other for promoting biotechnology in India. Some of these
centres are: NDRI—National Dairy Research Institute, Karnal,
Haryana

i. CDRI—Central Drug Research Institute, Lucknow, U.P.

ii. IVRI—Indian Veterinary Research Institute, Izatnagar. U.P. CFTRI
—Central Food and Technological Research Institute, Mysore

iii. CIMAP Central Institute of Medicinal and Aromatic Plants,

Lucknow, U.P.

iv. IITs of Kanpur, Madras, Bombay, New Delhi.

v. NBPGR—National Bureau of Plant Genetic Resources, New Delhi

Gene Therapy
Gene therapy in most simple words is the use of a gene to cure a
disease. There are a number of genetic diseases or acquired disorders
that may have occurred due to specific mutations in genes. Such
disorders may be corrected by replacing the defective gene by a normal
healthy gene.
This strategy of correcting the diseases is termed as gene therapy. So,
the gene therapy may be defined as the introduction of normal
functional gene in the defective cells of a patient to correct a genetic
or acquired disorder. The process of introduction of gene into the
appropriate cell of patient is called as the gene delivery.

During 1940s it was discovered that a gene from one bacterial strain
could be transferred into another strain and also that gene could be
expressed in another strain successfully. This discovery made the
researchers to think about the possibility that human genetic
disorder can be corrected in an analogous manner.

Introduction of a normal (therapeutic) gene into a cell having

defective gene, results into the correction of disorder because the
transferred gene provides the normal required gene product and this
whole strategy is termed as the gene therapy.
A number of human diseases have been targeted for gene
therapy. Some of these are:
A. Genetic diseases like Cystic Fibrosis, Haemophilia-A, B,
Phenylketonuria, Severe combined Immunodeficiency Disease
(SCID), etc.

B. Acquired diseases like Rheumatoid arthritis, AIDS, Cancer, etc.

While performing gene therapy, one of the two strategies can be
followed for gene delivery.

These two strategies are given below:

(i) In-vivo:
In this strategy, the normal therapeutic gene is introduced directly
into the target cell of patient.

(ii) Ex-vivo:
In this type of approach, the cells are isolated, cultured in-vitro and
then the normal gene is introduced into these cells. Such transformed
cells are then transplanted into the patient. Gene therapy can be done
at two levels for disease-correction, either at the embryo level called
as embryo therapy in which inheritance is also altered, or it can be
done at the patient level which is called as the patient therapy.

Types of Gene Therapy:

There are mainly two types of gene therapies; these are somatic gene
therapy and germ line gene therapy.

(a) Somatic Gene Therapy:

In this type, the therapeutic gene is introduced in the somatic cells of
the patient. The effect so produced is not heritable. This approach is
being used for trials made to treat cancer and blood disorders mainly.

(b) Germ line Gene Therapy:

In this type of gene therapy, the functional normal genes are
introduced into the germ cells like sperm and eggs to correct the
disorder. The changes produced by such approach are heritable and
thus are passed to the next generations.

Bio Fertilizers
Bio fertilizers are described as the microorganisms which are utilized
as fertilizers for plants as they enhance the availability of nutrients
like Nitrogen (N) and Phosphorus (P) to the plants. Another term
used for bio fertilizers is Microbial Inoculants. A number of biological
agents are being employed at large scale for the commercial
preparation of bio fertilizers which include algae, bacteria and fungi.

So, we may define bio fertilizers as the microbial inoculants of

bacteria, algae and fungi which increase the availability of nutrients
like N, P to the plants and thus result into benefit of plants. The
importance of bio fertilizers has been realized now and therefore a lot
of efforts are being made by the government as well as private sector
to encourage the use of bio fertilizers.

The microbial inoculants/bio fertilizers serve following

advantageous aspects:
(i) These are economical.

(ii) Unlike chemical fertilizers, they are environment friendly.

(iii) Bio fertilizers do not damage the soil texture.

(iv) They not only provide nutrition to the plants but also help in
enhancing the plant growth and yield.
On the basis of the type of nutrient provided by the bio
fertilizers, they can be categorized as follows:
(a) Nitrogen Bio fertilizers:
These are the microbial inoculants which enhance the availability of
nitrogen by fixation of atmospheric nitrogen. Examples of this
category include Rhizobium, Azospirillum, Cyanobacterium, etc.

(b) PhosphaticBiofertilizers:
These are the bio fertilizers which are responsible to increase the
availability of nutrient phosphorus to the plant by solubilizing the
soil phosphorus. Bacteria like Thiobacillus, Bacillus, etc. are
important examples of such category.

Some important microorganisms which are used

commercially as bio-fertilizers are enlisted below:

For large scale production of bio-fertilizers, it is choose the efficient

strains for N2-fixation and/or P-solubilization. To ensure the longevity
of bio-fertilizers, their storage and distribution systems must be
proper. In India, there is a continuous progress of bio-fertilizers
exploitation.
A number of private industries are also involved in manufacturing of
bio fertilizers. Government has also prepared a range of standards
regarding the maintenance and quality of bio-fertilizers. A National
Bio-fertilizer Development Centre is located at Ghaziabad in U.P.
which functions for the quality check and development of bio-
fertilizers in India.

Gene Bank
A gene bank is a facility where the genetic material is stored in the
form of seeds or plant parts at low temperatures. It serves as an
efficient method to store the germplasm of wild as well as cultivated
plants and therefore it helps in conserving the vanishing genetic-
diversity.

A gene bank is actually like a compartmentalized cold storage where

the genetic material is stored under controlled conditions of the
temperature and humidity for their germplasm conservation.
Conventionally ‘seeds’ are preferred as the material for germplasm
conservation.
The principle of a gene bank is that the dehydrated seeds can retain
their viability for a longer period of time if stored in cold conditions.

For a long-term storage usually a temperature ranging between 0-

18°C is applied. However, cryopreservation has made it more easy to
store seeds in viable condition for even longer durations of time. In
cryopreservation, the genetic material is stored in liquid nitrogen
having a very low temperature of -196 C.

Along with conserving the original genetic diversity, gene banks also
make the genetic material available as raw material to the breeders
and biotechnologists.

A few of the important gene banks are located at Vavilov Institute

(Russia), National seed storage laboratory (Fort Collins, USA),
International Rice Research Institute (Philippines), National Bureau
of Plant Genetic Resources (New Delhi) and Royal Botanic Garden
(Kew).

BIBLIOGRAPHY
The following books were used in the completion of this
project:

 Biology Lab Manual class 12.

 NCERT Textbook class 12.

Also, the following websites were consulted for relevant

material:

www.wikipedia.org
www.google.com
www.yahoo.com