CHANDRA SEKHAR ACADEMY

PURI-KONARK MARINE DRIVE ROAD,

BALIGHAI, PURI, ODISHA

2018 – 2019

AN INVESTIGATORY PROJECT
ON
APPLICATIONS OF BIOTECHNOLOGY

SUBMITTED BY
ASWESHA SARANGI
CLASS – XII
SUBJECT – BIOLOGY

SUBMITTED TO
PRIYAMBADA PATTANAIK
 Contents
1. Introduction
2. History
3. Biotechnology in Agriculture
4. Genetically Modified Crops

 RNA Interference (RNAi)

 Bt toxin
5. Bt cotton
6. Biotechnology in Medicine
7. Genetically engineered insulin (Humulin)
8. Gene therapy
9. Conclusion
10. Bibliography
 AKNOWLEDGEMENT
I am overwhelmed in all humbleness and gratefulness to acknowledge my depth to all
those who have helped me to put these ideas, well above the level of simplicity and into
something concrete.

I would like to express my special thanks of gratitude to my biology teacher,

PRIYAMBADA PATTANAIK as well as our Principal Miss. Amita Pattnaik who gave
me the golden opportunity to do this wonderful project on the topic “Applications of
Biotechnology”, which also helped me in doing a lot of research and I came to know
about so many new things. I am really thankful to them.

Any attempt at any level can’t be satisfactorily completed without the support and
guidance of my Parents and Friends who helped me a lot in gathering different
information, collecting data and guiding me from time to time in making this project,
despite of their busy schedules, they gave me different ideas in making this project
unique. I am thankful to them too.

I am making this project not only for marks but to also increase my knowledge...
Thanking you

ASWESHA SARANGI
CLASS - XII
 CERTIFICATE
This is to certify that ASWESHA SARANGI of class XII
of CHANDRA SEKHAR ACADEMY has successfully
completed the investigatory project on the topic
“APPLICATIONS OF BIOTECHNOLOGY” under the
guidance of PRIYAMBADA PATTANAIK during the
session 2018-19 in the partial fulfilment of Biology
Practical Examination conducted by CENTRAL BOARD
OF SECONDARY EDUCATION (AISSCE).

Teacher’s Sign Student’s Sign

Principal’s Sign External’s Sign

 INTRODUCTION
What is Biotechnology?
Biotechnology is the use of living systems and organisms to develop or make
products, or "any technological application that uses biological systems, living
organisms or derivatives thereof, to make or modify products or processes for
specific use. At its simplest, biotechnology is technology based on biology
biotechnology harnesses cellular and bio molecular processes to develop
technologies and products that help improve our lives and the health of our planet.
We have used the biological processes of microorganisms for more than 6,000
years to make useful food products, such as bread and cheese, and to preserve dairy
products.

Modern biotechnology provides breakthrough products and technologies to combat

debilitating and rare diseases, reduce our environmental footprint, feed the hungry,
useless and cleaner energy, and have safer, cleaner and more efficient industrial
manufacturing processes.
Biotech is helping to heal the world by harnessing nature's own toolbox and using
our own genetic makeup to heal and guide lines of research by:
• Reducing rates of infectious disease
• Saving millions of children's lives
• Changing the odds of serious, life-threatening conditions affecting millions
around the world
• Tailoring treatments to individuals to minimize health risks and side effects
• Creating more precise tools for disease detection
• Combating serious illnesses and everyday threats confronting the developing
world.

BIOTECHNOLOGY IN EARLY DAYS

Throughout the history of agriculture, farmers have inadvertently altered the
genetics of their crops through introducing them to new environments and breeding
them with other plants - one of the first forms of biotechnology.

These processes also were included in early fermentation of beer.

In brewing, malted grains (containing enzymes) convert starch from grains into
sugar and then adding specific
yeasts to produce beer. In this
process, carbohydrates in the grains
were broken down into alcohols
such as ethanol. Fermentation
was also
used in this time period to
produce leavened bread. Although
the process of fermentation was not
fully understood until Louis
Pasteur's work in 1857, it is still the
first use of biotechnology to
convert a food source into another form.

For thousands of years, humans have used selective breeding to improve production
of crops and livestock to use them for food. In selective breeding, organisms with
desirable characteristics are mated to produce offspring with the same
characteristics. For example, this technique was used with corn to produce the
largest and sweetest crops.

Biotechnology has also led to the development of antibiotics. In 1928, Alexander

Fleming discovered the mould Penicillium.
 BIOTECHNOLOGY IN AGRICULTURE
Genetically Modified Crops

Genetically modified crops or “GM

crops” or “biotech crops” are plants used in
agriculture, the DNA of which has been
modified with genetic engineering
techniques. In most cases the aim is to
introduce a new trait to the plant which does
not occur naturally in the species.

Examples in food crops include resistance to certain pests, diseases, stressful

environmental conditions, resistance to chemical treatments, reduction of spoilage,
or improving the nutrient profile of the crop. Examples in non-food crops include
production of pharmaceutical agents, bio fuels, and other industrially useful goods,
as well as for bioremediation.

Plants and crops with GM traits have been tested more than any other crops—with
no credible evidence of harm to humans or animals. In fact, seeds with GM traits
have been tested more than any other crops in the history of agriculture – with no
credible evidence of harm to humans or animals.

Genetic modifications have:

1. Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).

2. Reduced reliance on chemical pesticides (pest resistant crops).

3. Helped to reduce post harvest losses & enhanced the nutritional value of the
foods.

RNA Interference (RNAi)

RNA interference (RNAi) is a method of blocking gene function by inserting short

sequences of ribonucleic acid (RNA) that match part of the target gene’s sequence,
thus no proteins are produced. RNAi has the potential to become a powerful
therapeutic approach toward targeted and personalized medicine. RNAi has
provided a way to control pests and diseases, introduce novel plant traits and
increase crop yield. Using RNAi, scientists have developed novel crops such as
nicotine-free tobacco, non-allergenic peanuts, decaffeinated coffee, and nutrient
fortified maize among many others.

Mechanism of RNA interferences as understood is that it comes into play when a

double stranded RNA is introduced either naturally or artificially in a cell. An endo
ribonuclease enzyme cleaves the long dsRNA into small pieces of RNA. The small
pieces could be mi RNA or si RNA depending upon the origin of long dsRNA i.e.
endogenous or exogenous
respectively. A
double stranded RNA
may be generated by either
RNA dependent RNA
polymerase or bidirectional
transcription of transposable
elements or physically
introduced.

There are several

opportunities for the
applications of RNAi in crop
science for its improvement
such as stress tolerance and
enhanced nutritional level.This knockdown technology may be useful in inducing
early flowering, delayed ripening, delayed senescence, breaking dormancy, stress-
free plants, overcoming self-sterility, etc.

RNA interference (RNAi) has recently been demonstrated in plant parasitic

nematodes. It is a potentially powerful investigative tool for the genome-wide
identification of gene function that should help improve our understanding of plant
parasitic nematodes. RNAi should help identify gene and, hence, protein targets for
nematode control strategies. Prospects for novel resistance depend on the plant
generating an effective form of double-stranded RNA in the absence of an
endogenous target gene without detriment to itself. These RNA molecules must
then become available to the nematode and be capable of ingestion via its feeding
tube. If these requirements can be met, crop resistance could be achieved by a plant
delivering a dsRNA that targets a nematode gene and induces a lethal or highly
damaging RNAi effect on the parasite.
Bt Cotton
Bt cotton is a genetically modified organism (GMO) cotton variety, which produces an
insecticide to bollworm. Strains of the bacterium Bacillus thuringiensis produce over
200 different Bt toxins, each harmful to different insects. Most notably, Bt toxins are
insecticidal to the larvae of moths and butterflies, beetles,
cotton bollworms and ghtu flies but are harmless to other
forms of life. The gene coding for Bt toxin has been
inserted into cotton as a transgene, causing it to produce
this natural insecticide in its tissues. In many regions, the
main pests in commercial cotton are lepidopteran larvae,
which are killed by the Bt protein in thegenetically
modified cotton they eat. This eliminates the need to use
large
amounts of broad-spectrum insecticides to kill
lepidopteran pests. This spares natural insect predators in
the farm ecology and further contributes to non insecticide
pest management.

Bt cotton is ineffective against many cotton pests

such as plant bugs, stink bugs, and aphids;
depending on circumstances it may be desirable to
use insecticides in prevention.

Mechanism:

Bt cotton was created through the addition of

genes encoding toxin crystals in the Cry group of endotoxin. When insects attack and eat
the cotton plant the Cry toxins are dissolved due to the high pH level of the insects
stomach. The dissolved and activated Cry molecules bond to cadherin-like proteins on
cells comprising the brush border molecules. The epithelium of the brush border
membranes separates the body cavity from the gut whilst allowing access for nutrients.
The Cry toxin molecules attach themselves to specific locations on the cadherin-like
proteins present on the epithelial cells of the midge and ion channels are formed which
allow the flow of potassium. Regulation of potassium concentration is essential and, if left
unchecked, causes death of cells. Due to the formation of Cry ion channels sufficient
regulation of potassium ions is lost and results in the death of epithelial cells. The death of
such cells creates gaps in the brush border membrane.
Advantages:
Bt cotton has several advantages over non Bt cotton. The important advantages of Bt
cotton are briefly :

• Increases yield of cotton due to effective control of three types of bollworms, viz.
American, Spotted and Pink bollworms.

• Insects belonged to Lepidoptera (Bollworms) are sensitive to crystalline endotoxic

protein produced by Bt gene which in turn protects cotton from bollworms.

• Reduction in pesticide use in the cultivation of Bt cotton in which bollworms are

major pests.

• Reduction in the cost of cultivation and lower farming risks.

• Reduction in environmental pollution by the use of insecticides rarely.

• Bt cotton exhibit genetic resistance or inbuilt resistance which is a permanent type

of resistance and not affected by environmental factors. Thus protects crop from
bollworms.

• Bt cotton is ecofriendly and does not have adverse effect on parasites, predators,
beneficial insecticides and
organisms present in soil.

• It promotes multiplication
of parasites and predators
which help in controlling the
bollworms by feeding on
larvae and eggs of bollworm.

• No health hazards due to rare

use of insecticides.

• Bt cotton are early in

maturing as compared to non
Bt cotton.
Disadvantages:
Bt cotton has some limitations

• High cost of Bt cotton seeds as compared to non Bt cotton seeds.

• Effectiveness up to 120 days, after that the toxin producing efficiency of the Bt
gene drastically reduces.

• Ineffective against sucking pests like jassids, aphids, whitefly etc.

Bt cotton in India:
Bt cotton is supplied in India's Maharashtra state by the agribiotechnology company,
Mahyco, as the distributor.

The use of Bt cotton in India has grown exponentially since its introduction. Recently
India has become the number one global exporter of cotton and the second largest cotton
producer in the world. India has bred Bt-cotton varieties such as Bikaneri Nerma and
hybrids such as NHH-44, setting up India to benefit now and well into the future.

India’s success has been subject to scrutiny. Monsanto's seeds are expensive and lose
vigour after one generation, prompting the Indian Council of Agricultural Research to
develop a cheaper Bt cotton variety with seeds that could be reused. The cotton
incorporated the cry1Ac gene from the soil bacterium Bacillus thuringiensis (Bt), making
the cotton toxic to bollworms. In parts of India cases of acquired resistance against Bt
cotton have occurred.

The state of Maharashtra banned the sale and distribution of Bt cotton in 2012, to promote
local Indian seeds, which demand less water, fertilizers and pesticide input, but lifted the
ban in 2013.
India approved Bt cotton in 2002; now it accounts for 92% of all Indian cotton. Average
nationwide cotton yields went from 302 kg/ha in the 2002/3 season to a projected 481
kg/ha in 2011/12 — up 59.3% overall. This chart shows the trends in yields, which took
off after Bt was introduced in 2002. The graphs also show that — and here comes ugly
fact— in the last 4 years, as Bt has risen from 67% to 92% of India’s cotton, yields have
dropped steadily.
 BIOTECHNOLOGY IN MEDICINE
Genetically Engineered
Insulin (Humulin)
Insulin is a peptide hormone produced
by beta cells in the pancreas of various
organisms including human beings. It
regulates
the metabolism of carbohydrates an d
fats by promoting the absorption of
glucose from the blood to skeletal muscles and fat tissue and by causing
fat to be stored rather than used for energy. Insulin also inhibits the production of
glucose by the liver.

Structure:

Insulin is composed of two different

types of peptide chains. Chain A has 21
amino acids and Chain B has 30 amino
acids. Both chains contain alpha helices
but no beta strands. There are 3
conserved disulfide bridges which help
keep the two chains together.

Need of Genetically Engineered Insulin:

The original form of the

wonder cure for diabetes,
these were once the only
type of insulin available,
but are now rarely used.
Animal insulin was
originally made from
ground-up animal pancreas tissue, and then later was extracted from
healthy animals
(slaughtered pigs & cows).
One of the problems with animal insulin was antibody issues. The body identifies them
and tries to reject them.

Humulin:

Biosynthetic "human" insulin is now manufactured for widespread clinical use using
genetic engineering techniques using recombinant DNA technology, which the
manufacturers claim reduces the presence of many impurities, although there is no clinical
evidence to substantiate this claim. Eli Lilly marketed the first artificial insulin,
Humulin, in 1982.

Humulin production method is as follows:

1. DNA coding for A and B polypeptide chains of insulin are chemically synthesised
a in the lab. Sixty three nucleotides are sequenced to produce A chain of insulin
and ninety nucleotide long DNA designed to produce B chain of insulin, plus
terminator codon is added at the end of each chain sequence. Anti-codon for
methionine is added at the beginning of the sequence to distinguish humulin from
the other bacterial proteins.

2. Chemically synthesized A and B chain DNA sequence are inserted into one of the
marker gene which are present in the plasmid vector. Genes are inserted into the
plasmid with the help of enzymes known as endonuclease and ligase.

3. The vector plasmids with the insulin gene are then introduced into the E. coli
bacterial cell. These cells are then allowed to replicate by mitosis, along with the
bacterial cell recombinant plasmid also gets replicated producing the human
insulin.

4. A and B polypeptide chains of insulin are then extracted and purified from the
fomenters in the lab. High-Performance Liquid Chromatography (HPLC) is used
to get 100% pure humulin from the mixture of proteins.

5. The A and B polypeptide chains of insulin are mixed together and connected with
each other by disulphide bond, forming the Humulin or synthetic human insulin.
Advantages & Disadvantages of
Humulin:

Humulin is the one and only human

protein produced in the bacteria with
identical chemical structure to that of
the natural human insulin.
Administration of humulin reduces the possibility of antibody production and
inflammatory response in diabetic patients. Major difficulty is the extraction of humulin
from a mixture of host proteins present in the fermentation broth.

Now days to overcome this extraction problem synthetic human insulin are produced in
the yeast cell instead of E. coli using the same procedure. As yeast is Eukaryotes they
secrete the whole humulin molecule with perfect three dimensional structures, reducing
the need for complex and time consuming purification methods.
Now most of the diabetic patients are treated with synthetic human insulin. Small group of
patients claim that episodes of hyperglycaemic complications have been increased after
shifting from animal origin insulin to humulin. No study till date shows the difference
between the frequency of hyperglycaemic complications in patient using humulin
(synthetic human insulin) and animal origin insulin.

Gene Therapy
Gene therapy is the therapeutic delivery of nucleic acid polymers into a patient's
cells as a drug to treat disease. Gene therapy is an experimental technique that uses
genes to treat or prevent disease. In the future, this technique may allow doctors to
treat a disorder by inserting a
gene into a patient’s cells
instead of using drugs or
surgery. Researchers are
testing several approaches to
gene therapy, including:

• Replacing a mutated
gene that causes
disease with a healthy
copy of the gene.

• Inactivating, or
“knocking out,” a mutated gene that is functioning improperly.

• Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases
(including inherited disorders, some types of cancer, and certain viral infections),
the technique remains risky and is still under study to make sure that it will be safe
and effective. Gene therapy is currently only being tested for the treatment of
diseases that have no other cures. It should be noted that not all medical procedures
that introduce alterations to a patient's genetic makeup can be considered gene
therapy. Bone marrow transplantation, and organ transplants in general have been
found to introduce foreign DNA into patients. Gene therapy is defined by the
precision of the procedure and the intention of direct therapeutic effects.

Gene therapy was conceptualized in 1972, by authors who urged caution before
commencing human gene therapy studies.

The first attempt, albeit an unsuccessful one, at gene therapy (as well as the first
case of medical transfer of foreign genes into humans not counting organ
transplantation) was performed by Martin Cline on 10 July 1980. Cline claimed
that one of the genes in his patients was active six months later, though he never
published this data or had it verified and even if he is correct, it's unlikely it
produced any significant beneficial effects treating beta-thalassemia.

The first germ line gene therapy consisted of producing a genetically engineered
embryo in October 1996. The baby was born on July 21, 1997 and was produced
by taking a donor's egg with healthy mitochondria, removing its nuclear DNA and
filling it with the nuclear DNA of the biological mother - a procedure known as
cytoplasmic transfer.

This procedure was referred to sensationally and somewhat inaccurately in the

media as a "three parent baby", though mtDNA is not the primary human genome
and has little effect on an organism's individual characteristics beyond powering
their cells.

Gene therapy is a way to fix a genetic problem at its source. The polymers are
either expressed as proteins, interfere with protein expression, or possibly correct
genetic mutations.

The most common form uses DNA that encodes a functional, therapeutic gene to
replace a mutated gene. The polymer molecule is packaged within a "vector",
which carries the molecule inside cells.

The first commercial gene therapy, Gendicine, was approved in China in 2003 for
the treatment of certain cancers. In 2011 Neovasculgen was registered in Russia as
the first-in-class gene-therapy drug for treatment of peripheral artery disease,
including critical limb ischemia. In 2012 Glybera, a treatment for a rare inherited
disorder, became the first treatment to be approved for clinical use in either Europe
or the United States after its endorsement by the European Commission.

ADA deficiency is one form of SCID (severe combined immunodeficiency), a

disorder that affects the immune system. ADA deficiency is very rare, but very
dangerous, because a malfunctioning immune system leaves the body open to
infection from bacteria and viruses.

The disease is caused by a

mutation in a gene on
chromosome
20. ADA deficiency is inherited
in an autosomal recessive
manner. The gene codes for the
enzyme adenosine deaminase
(ADA). Without this enzyme, the
body is unable to break down a
toxic substance called
deoxyadenosine. The toxin
builds up and destroys infection-
fighting immune cells
called T and B lymphocytes.
Because ADA deficiency affects the
immune system, people who have the disorder are more susceptible to all kinds of
infections, particularly those of the skin, respiratory system, and gastrointestinal
tract. They may also be shorter than normal. Sadly, most babies who are born with
the disorder die within a few months.

Treatments of ADA Deficiency includes:

• bone marrow transplant

• gene therapy

• ADA enzyme in PEG vehicle

On September 14, 1990, the first gene therapy to combat this disease was
performed by Dr. William French Anderson on a four-yearold girl, Ashanti
DeSilva, at the National Institutes of Health, Bethesda, Maryland, U.S.A.
 CONCLUSION
Biotechnology is the new wonder of science. It is truly multidisciplinary in nature
and it encompasses several disciplines of basic sciences and engineering. The
Science disciplines from which biotechnology draws heavily are microbiology,
chemistry, biochemistry, genetics, molecular biology, immunology, cell and tissue
culture and physiology. On the engineering side it leans heavily on process
chemical and biochemical engineering since large scale cultivation of
microorganisms and cells, their downstream processing are based on them. It
comes to us as a great blessing...

Biotechnology utilizes the technique called genetic engineering or recombinant

DNA technology where a microorganism is isolated; its genetic material is cut,
manipulated, sealed, again inserted in an organism and allowed to grow in a
suitable environment under controlled conditions to get the desired product. It
looks easy but is a very tedious job and it takes years for a research to achieve its
goal.
Like every other thing, biotechnology too has some harmful impacts:
1. Genetic engineering is a very vital part of biotechnology and the cost of
transferring genes from one species to another is very expensive, which
requires a huge amount of capital investment. The cost of producing
genetically- modified plants and animals are sky- rocketing and the
duration of return are also not predictable.
2. Genetic engineering crosses boundaries of reproduction by crossing genes of
species that are completely unrelated; hence giving rise to hazardous results as
well as also increasing the risk of harming multiple species.
3. When genetic material from certain viruses is used in the production of
transgenic crops, there are chances that these virus genes will combine with
crop genes to produce more destructive viruses. The consumption of such crops
is hazardous to human health and can cause several life- threatening ailments.
It can also result in cancer, often malignant as well.
4. Biotechnology also poses a number of environmental threats. Genetically
modifies crops often infect monarch butteries and other insect species.

The applications of biotechnology are so broad, and the advantages so compelling,

that virtually every industry is using this technology. Developments are underway
in areas as diverse as pharmaceuticals, diagnostics, textiles, aquaculture, forestry,
chemicals, household products, environmental cleanup, food processing and
forensics to name a few. Biotechnology must continue to be carefully regulated
so that the maximum benefits are received with the least risk.
 Bibliography
 http://en.wikipedia.org/biotechnology
 http://en.wikipedia.org/insulin
 http://www.genewatch.org/sub-568238
 http://en.wikipedia.org/humulin
 http://www.biotecharticles.com/Others-Article/Human-Insulin-and-
Recombinant-DNA-Technology-70.html
https://isaaa.org/resources/publications/pocketk/34/default.asp
 http://www.sciencedirect.com/
https://en.wikipedia.org/wiki/Gene_therapy
 https://en.wikipedia.org/wiki/Adenosine_deaminase_deficiency
 http://www.diabetes.co.uk/insulin/animal-insulin.htmlBiology
textbook (N.C.E.R.T) Class 12th