Investigatory Project

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 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, Mrs Tapswani
as well as our Principal 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.

Abhinash Panda
XII
 Certificate
This is to certify that Abhinash Panda of class XII of
SANKALP SENIOR SECONDARY SCHOOL has successfully
completed the investigatory project on the topic
“APPLICATIONS OF BIOTECHNOLOGY” under the guidance
of MRS. TAPSWANI (H.O.D of Biology)during the session
2019-20 in the partial fulfilment of Biology Practical
Examination conducted by CENTRAL BOARD OF
SECONDARY EDUCATION (AISSCE).

Mrs. Tapswani 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 us.

At its simplest, biotechnology is technology based on biology biotechnology harnesses

cellular and bio molecular processes to develop technology 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 breadand
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.
 History
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 grain
beer. In this process, carbohydrates in the grains were broken down into alcohol such as
ethanol. Later other cultures produced the process of lactic acid fermentation which
allowed for preservation of other forms of food such as soya sauce.

Fermentation was also used in this time period to produce leavened bread. Although the
process of fermentation was not fully understood until Louis Pasteur 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. His work led to the purification of the antibiotic
compound formed by the mould by Howard Florey, Ernst Boris Chain and Norman Heatley
- to form what we today know as penicillin. In 1940, penicillin became available for
medicinal use to treat bacterial infections in humans.

The field of modern biotechnology is generally thought of as having been born in 1971 when
Paul Berg's experiments in gene splicing had early success. Herbert W. Boyer and Stanley
N. Cohen significantly advanced the new technology in 1972 by transferring genetic
material into a bacterium, such that the imported material would be reproduced.
 Biotechology in Medicine
Genetically Engineered Insulin(Humilin)

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.
Except in the presence of the metabolic disorder diabetes mellitus and metabolic
syndrome, insulin is provided within the body in a constant proportion to remove excess
glucose from the blood, which otherwise would be toxic. When blood glucose levels fall
below a certain level, the body begins to use stored glucose as an energy source through
glycogenolysis, which breaks down the glycogen stored in the liver and muscles into
glucose, which can then be utilized as an energy source. As a central metabolic control
mechanism, its status is also used as a control signal to other body systems (such as amino
acid uptake by body cells). In addition, it has several other anabolic effects throughout the
body. When control of insulin levels fails, diabetes mellitus can result.

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. Insulin
can also form dimers in solution due to the hydrogen bonding between the B chains. The
dimers can further interact to form hexamers due to interaction between hydrophobic
surfaces. This scene highlights the hydrophobic and polar parts of an insulin monomer at a
pH of 7.

A number of insulin variants have been made to favor either the monomeric or hexameric
form. Deletion of the five C terminal residues of the B chain creates a monomer only form.
This portion of the B chain is involved in hydrogen bonds between the B chain of one
monomer and the A and B chain of another monomer.
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 animal .

The metabolism of cows and pigs was close enough to human metabolism that their animal
insulin also worked well in human bodies. Beef insulin has 3 differences from human; pork
insulin has 1 difference from human. The use of a mixture of beef and pork insulin was also
possible. It has been shown that human insulin is less immunogenic than animal insulin.
Porcine insulin is most similar to human insulin. The primary amino acid sequences of
bovine and porcine insulin differ from that of human insulin by three and one amino acid,
respectively. This greater dissimilarity between human and bovine insulin has been
postulated to be the explanation for the greater antigenicity of bovine insulin as compared
with porcine insulin

One of the problems with animal insulin was antibody issues. The body identifies them
and tries to reject them. Pork insulin differs by 1 amino acid and beef insulin by 3 amino
acids, so the body's immune system can sometimes recognize them as foreign.
Immunological complications of insulin therapy have been evident since animal insulin
became available for the treatment of diabetes mellitus in 1922. In insulin-allergic patients
treated with conventional insulin preparations, the insulin-specific IgE values are often 10-
to 20-fold higher than in patients without allergy. It has been shown that human insulin is
less immunogenic than animal insulin. Porcine insulin is most similar to human insulin.
Crossreactivity between human insulin and insulin of animal origin has been reported. A
major problem is the cross-reactivity that occurs between anti-insulin antibodies and the
various animal and human insulin preparations in patients presenting with allergy to animal
insulin.

The usage of animal insulin has so greatly declined in modern times that they have largely
been withdrawn from the market. Newly diagnosed diabetics are typically given or
Genetically Engineered human insulin.

What is “Proinsulin”?

Proinsulin is the prohormone precursor to insulin made in the beta cells of the islets of
Langerhans, specialized regions of the pancreas. Proinsulin is synthesized on membrane
associated ribosomes found on the rough endoplasmic reticulum, where it is folded and
its disulfide bonds are oxidized. It is then transported to the Golgi apparatus where it is
packaged into secretory vesicles, and where it is processed by a series of proteases to
form mature insulin. Mature insulin has 35 fewer amino acids; 4 are removed altogether,
and the remaining 31 form the C-peptide. The C-peptide is abstracted from the center of
the proinsulin sequence; the two other ends (the B chain and A chain) remain connected
by disulfide bonds.

When insulin was originally purified from bovine or porcine pancreata, all the proinsulin
was not fully removed.When some people used the insulins, the proinsulin may
have caused the body to react with a rash, to resit the insulin, or even to make dents.
or lumps in the skin at the place where the insulin was injected. This can be described as
an iatrogenic injury due to slight differences between the proinsulin of different species.
Since the late 1970s, when highly purified porcine insulin was introduced, and the level of
insulin purity reached 99%, this ceased to be a significant clinical issue. The main challenge
for production of insulin using rDNA techniques was getting insulin assembled into
mature form.

Humulin:

Humulin was the first medication produced using modern genetic engineering techniques
in which actual human DNA is inserted into a host cell (E. coli in this case). 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.

20. ADA
The disease is caused by a mutation in a gene on chromosome 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