Investigatory Project

Aswesha sarangi
Mrs
XII Sci. B
(H.O.D Biology)

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, Mr. Sandeep
Kulshesthra as well as our Principal Mrs. Nidhi Bhatia 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
Subhag Singh
XII Sci. B
 Certificate
This is to certify that SUBHAG SINGH of class XII SCI.B of
GYAN DEEP SHIKSHA BHARATI has successfully
completed the investigatory project on the topic
“APPLICATIONS OF BIOTECHNOLOGY” under the
guidance of MR. SANDEEP KULSHESTHRA (H.O.D.
Biology) during the session 2015-16 in the partial
fulfilment of Biology Practical Examination conducted by
CENTRAL BOARD OF SECONDARY EDUCATION (AISSCE).
___________________
___________________
Mr. Sandeep Kulshesthra External
Examiner
(H.O.D Biology) (C.B.S.E)

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 is enabling these industries to make new or better products, often
with greater speed, efficiency and flexibility. 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_deficie

ncy
http://www.diabetes.co.uk/insulin/animal-insulin.html
Biology textbook (N.C.E.R.T) Class 12th

Contents
 Introduction  History  Biotechnology in
Agriculture
• Genetically Modified Crops
• RNA Interference (RNAi)
 Bt toxin  Bt cotton  Biotechnology in
Medicine  Genetically engineered insulin
(Humulin)  Gene therapy  Conclusion 
Bibliography