Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

APPLICATIONS OF BIOTECHNOLOGY

The applications of biotechnology include the following fields:

 Therapeutics
 Diagnostics
 Genetically modified crops
 Food processing
 Bioremediation
 Waste treatment
 Energy production, etc.

Three critical research areas of biotechnology:

1. Developing best catalyst
a. Catalyst for biotechnology work include improved strains of organisms such as
microbes
b. Enzymes such as REE, ligase, etc.
2. Creating optimal conditions for the catalyst to act
a. Such as developing better growth medium for growing microorganisms and other
hosts
3. Developing better downstream processing technologies
a. The best quality product development depends on downstream processing
employed to purify the proteins or the products.

Applications of Biotechnology

Agriculture Medicine Transgenic animal

Recombinant Proteins Therapeutics Molecular Diagnosis

Applications in Agriculture

 Genetically Modified Organisms (GMOs): organisms which are genetically engineered for
human benefit.
 Genetic modifications of plants have following advantages:
1. Abiotic stress tolerant crops
a. GM plants can tolerate extreme cold, draught, heat, high salt conc. in soil, etc.
2. Reduced reliance on chemical pesticides
a. Reduces the side effects of pollution caused by chemical pesticides
b. Insect resistant and pest resistant plants were developed
3. Reduced post harvest losses
a. GM plants have higher shelf life
b. They are resistant to microbial infections
4. Increased efficiency of mineral usage by plants
a. So that the soil fertility is not exhausted early
5. Enhanced nutritional value
a. So that food security can be ensured
b. Plants rich in vitamins, calcium, etc are created

Notes prepared by MRINAL CHOUDHURY Page 1 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

Bt Cotton – Insect Resistant Plant

 Cotton plants are often infected by various insects such as

o Lepidopterans, e.g. tobacco budworm, armyworm
o Coleopterans, e.g. beetles
o Dipterans, e.g. flies, mosquitoes

 Bacteria Bacillus thuringiensis contain cry genes – cryIAb, cryIAc and cryIIAb
 Cry genes produce insecticidal proteins effective against the cotton infecting insects.
 However, they are produced in inactive form inside bacteria and hence do not effect them.
 Genetic engineering of cotton plants: the process is as follows

Cry genes form Bacillus thuringiensis are isolated

↓
Genes are introduced into cotton plant cells using Ti plasmid
(Agrobacterium mediated transfer)
↓
Genetically modified plant cells ate cultured to produce genetically modified plants
↓
GM cotton plants produce inactive Cry toxin protein in its cells
↓
When insects feed on the cotton tissues, inactive toxin enters the insect gut
↓
In the alkaline pH of insect gut, inactive toxin becomes active toxic protein
↓
Toxin binds the midgut surface and produce pores in the gut epithelium
↓
The cells swell up and burst, killing the insect.

Pest Resistant plant

 Roots of tobacco plants are often infected by Meloidogyne incognita

 They reduce the plants roots’ absorbing capacity and thus reduce the yield.
 A novel strategy known as RNA interference or RNA Silencing is employed to make pest
resistant plants.

Notes prepared by MRINAL CHOUDHURY Page 2 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

 The process of RNA interference is as follows:

Usually source of RNA is Retrovirus infection or Transposons.

For pest resistance, an artificial pest specific DNA is constructed
↓
Pest specific DNA is introduced into the plant cells through Agrobacterium mediated transfer
↓
Transcription of the pest specific DNA from both the sides
produce a dsRNA
↓
Dicer protein acts on the dsRNA and cleaves it to form multiple
siRNAs (small interfering RNA)
↓
RISC (RNA induced silencing complex) binds to it the siRNAs to
form activated RISC
↓
Activated RISC* becomes single stranded which is
complementary to an important nematode mRNA
↓
When a nematode infects the plant, the RISC* encounters the
nematode mRNA and binds to it
↓
RISC* cleaves the nematode mRNA
↓
Thus nematode mRNA is silenced, i.e. cannot undergo translation
and leads to death of nematode

Recombinant Protein Production – Human Insulin (Humulin)

 30 therapeutic recombinant proteins are approved for human use, 12 are marketed in India.
 One of the most common diseases among all human is Diabetes.
 Since the time it was discovered that deficiency of hormone Insulin is one important reason
for its onset, different insulin molecules from various sources such as cow and pig have been
tried.
 Although, they were functional to some extent and induces immunogenic response in the
body.
 After the birth of genetic engineering, recombinant human insulin is being made in a
bacterial host.
 The first such recombinant human insulin is branded as Humulin
 It was manufactured by Eli Lilly and Co., and American company in 1983.
 Structure of normal in vivo Human Insulin

o INS gene at chromosome no 11 codes for human insulin.

Notes prepared by MRINAL CHOUDHURY Page 3 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

o Human insulin is translated as a fusion protein, Preproinsulin of Signal peptide, B

chain, C chain and A chain having a total of 110 amino acids.
 Signal peptide – 24 amino acids
 B chain – 30 amino acids
 C chain – 35 amino acids
 A chain – 21 amino acids
o Signal peptide directs the preproinsulin to golgi bodies for modifications
o C chain induces a folding of the preproinsulin
o Signal peptide is cleaved inside golgi and two disulfide bonds are formed between A
and B chains.
o C chain is cleaved and degraded
o Mature insulin has two chains A and B, having 51 amino acids and has a molecular
weight of 5808 Da.

 Production of recombinant protein Insulin

A and B chains are artificially

synthesized separately
↓
They were ligated into two separate
plasmids along with β-gal gene
↓
Recombinant plasmids were
transformed into E.coli
↓
Expression of insulin gene led to
formation of β-gal/insulin fusion
proteins inside the cells
↓
They were extracted after mass
production
↓
Treatment with cyanogens bromide
cleaved β-gal from A and B chain
polypeptides
↓
Mixing the purified A and B chains produced active Insulin molecule by formation of two
disulfide bonds

 Assembly of mature insulin by forming disulfide bonds between A and B chains is a

challenge, which often reduces the efficiency of the process.
 This challenge was overcome by another approach by Genetech
o A proinsulin cDNA was used as gene of interest and proinsulin was produced by the
same process as followed by Eli lilly
o But the presence of C chain in the final protein, induced the natural folding and
bonding of A and B chains
o C chain was cleaved by treating with carboxypeptidase and thus mature insulin was
formed.

Notes prepared by MRINAL CHOUDHURY Page 4 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

Gene Therapy

 Gene therapy literally means replacing the defective gene by a functional copy of the same.
 First gene therapy was successfully carried out for ADA-SCID in a 4 year old Sri Lankan girl
Ashanti de-Silva by Dr. William French Anderson at National Institute of Health, USA in
1990
 ADA-SCID: Adenosine Deaminase Severe Combined Immunodeficiency
o Adenosine is an enzyme coded by ada gene in chromosome 20
o It is expressed in the lymphocytes necessary for proper immune system development
o Its deficiency causes severyly compromised immune system
o Conventional treatment includes –
 Enzyme Replacement Therapy – not a permanent treatment procedure as
frequent injections of enzyme is necessary
 Bone Marrow Transplantation – not feasible always due to non availability of
matching donor
 Gene Therapy for ADA SCID

Patient lymphocytes are isolated and cultured

↓
The abnormal lymphocytes are treated with disarmed retrovirus carrying recombinant RNA of
normal ADA gene.
↓
Inside the cells recombinant RNA produce recombinant DNA by the action of enzyme reverse
transcriptase.
↓
Normal recombinant DNA (ADA gene) is integrated into the lymphocytes’ DNA forming
genetically modified lymphocytes
↓
GM lymphocytes are selected for successful transformation and cultured to increase their number
↓
GM lymphocytes are injected into patient blood stream
 Since the cell contain a copy of the normal gene, normal ADA is produced in the body
 Since lymphocytes are of patient origin, there is no problem of matching donor

 Limitation of the process:

Notes prepared by MRINAL CHOUDHURY Page 5 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

o Since lymphocytes have a fixed life span, GM lymphocytes need to regularly injected
into patient’s blood.
o This limitation may be removed by carrying out gene therapy at an early embryonic
stage so that all the cells of body receive the normal copy of the gene.

Molecular Diagnosis

 Treatment of a disease depends on its accurate and early detection/diagnosis.

 Conventional diagnosis is done by testing samples such as serum, urine, stool, sputum, etc.
 However, such diagnostic tools are not efficient enough for accurate and early diagnosis.
 Accurate and early diagnosis can be done by using molecules such as DNA and proteins.
 Use of molecules for diagnosis is known as Molecular Diagnosis
 Various techniques such as PCR, ELISA and Radioactive probe are used for molecular
diagnosis.

Molecular Diagnosis – PCR

 Many acute diseases such as AIDS and Cancer require early detection to be treated
efficiently.
 However, their diagnosis depends upon the quantity of infectious agents or amount of
mutations present in the body.
 Thus PCR is employed to detect the presence of bacteria or virus or mutations in DNA
 Process of detection
o Primers are used to amplify the pathogen DNA or
the mutated DNA
o If the pathogen is present even in very minute
quantity, its DNA is amplified
o During the electrophoresis, a band for pathogen
DNA is observed along with the host DNA.
 Types of PCR for diagnosis –
o Qualitative PCR – only presence or absence of a
pathogen is detected
o Quantitative PCR – quantity of pathogen or
mutation is detected.

Molecular Diagnosis – ELISA

 ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for

detecting and quantifying substances such as peptides, proteins, antibodies and hormones.
 Whenever, there is an infection, an antigenic determinant is present in the serum, which may
be detected by ELISA
 Else, when an antigen is present, immune system produces antibodies against it, such
antibodies can be detected by ELISA, which shows the presence of antigen.

Notes prepared by MRINAL CHOUDHURY Page 6 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

 The basic process of ELISA is as follows:

A microtitre ELISA plate is taken having multiple small wells

(as in the pic.)
↓
The buffered serum where antigen is to be tested is taken in the
wells of microtitre plate
↓

Some time is allowed for the antigens to adhere to the wells

↓
A solution of non reacting protein such as Bovine Serum
Albumin is added to coat any surface on the plate which is
uncoated with antigen ↓
↓

An antibody attached (linked) to an enzyme is added which

specifically binds to the antigen
↓

↓
A substrate is added for the enzyme which changes colour
when acted upon by the enzyme thus showing the presence of
antigen in the serum
↓
Qualitative Quantitative
estimate estimate

Appearance of color More is the quantity of antigen

indicates the presence of in the serum, more intense the
antigen color will be
↓ ↓
A positive test Can be estimated using
colorimeter

Molecular Diagnosis – Radio labelled Probe

 Probe: an artificially synthesized single stranded DNA or RNA sequence of variable length
(usually 100-1000 bases long).
 A radiolabelled probe has a radioactive substance coupled to it.
 The sequence of the probe is complementary to the target DNA sequence to be identified.
 This technique can be used for identifying the presence of mutations in a sample of tissues.
 A brief overview of the principle:
A radiolabelled probe is synthesized having sequence complementary to normal gene
sequence
↓
The DNA from tissue sample is exposed to Radiolabelled probe for hybridisation
↓
Hybridised sample is exposed to X ray film for detecting radioactive signals
↓

Notes prepared by MRINAL CHOUDHURY Page 7 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

Interpretation of observations:
 If the gene is not mutated, probe binds to it and gives a positive signal for radioactivity
 If the gene is mutated, its sequence changes, as a result, probe does not bind to it, and gives a
negative result

TRANSGENIC ANIMALS

 Transgene: a foreign gene that is introduced into a host cell

 Transgenic organisms: An organism produced by introducing a foreign gene into it

to study normal
physiology and
development
to study to study a
chemical human
toxicity disease and
new drug
TRANSGENIC trial
ORGANISMS

to produce
To study the useful
vaccine safety biological
products

 Model organism: an organism that has a physiology similar to human beings, which is used
to study human physiology or pathophysiology.
o Transgenic Model Organisms: they are produced by introducing a foreign gene
into a model organism which can produce a typical phenotype similar to human.
o Knock out model organism: a transgenic animal in which a specific gene is
inactivated by introducing another similar gene (having enough changes in sequences
to produce an abnormal copy of the gene product)
The introduced gene due to its homology with the host gene gets incorporated in the
place of host gene by replacing the same.

 Transgenic Model Organisms to study normal physiology and development of Human:

o Knock out mice models are made to inactivate specific genes similar to human and
study
 the function of the gene product
 its role in the normal physiology and development
 how the gene is regulated
o e.g. – effect of Insulin like Growth Factor, PDGF, etc.
 Transgenic Model Organisms to study Diseases and new Drug Effect:
o Knock out mice models are also made to deactivate certain genes responsible for
various single gene disorders and other genetic diseases.
o Such diseases can be studied to understand their pathophysiology and disease
progression
o Any new drug or other medical interventions developed for such diseases can be
tested in transgenic model organisms.

Notes prepared by MRINAL CHOUDHURY Page 8 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

o E.g. - model organisms are available for cancer, cystic fibrosis, rheumatoid arthritis,
Alzheimer’s disease, etc.
 Production of Biological Products:
o Various biological products have uses in medicine.
o But they are either difficult to extract from animal tissues or difficult or too expensive
to synthesize.
o Such products may be produced by genetic engineering as follows:

 The specific human gene is isolated from human DNA

 Unfertilised egg is obtained from a suitable donor
↓
Human gene is introduced into the egg by
microinjection
↓
The transgenic egg is introduced into the host female
organism (usually a surrogate goat or cow)
↓
The new offspring (transgenic) produced will carry
the human gene
↓
The transgenic adult will secrete the human protein in its
milk

o E.g. –
1. 1997, 1st transgenic cow, Rosie was born, produced 2.4 gm/ltr of α-lactalbumin
2. Human protein α-1-antitrypsin for treatment of emphysema
3. Tissue Plasminogen Activator from transgenic goat
4. Blood Clotting Factor VIII and IX from transgenic sheep

 Testing Vaccine Safety using Transgenic mice models:

o Since vaccine contain antigens although in weakened state, it cannot tested in humans
directly, so they are tested in animal models first
o Transgenic mice are replacing monkeys for testing the safety of new vaccine.
o E.g. – Polio Vaccine
 Testing safety of chemicals in hypersensitive transgenic models:
o Transgenic model organisms are made to be hypersensitive for specific toxic
chemicals so that even minute amount of toxic chemicals in foods or drugs or
cosmetics can be determined.
 Rapid Growth promotion in commercial animals:
o Transgenic animals are made by introducing growth promoting genes to induce faster
and better growth or increase in size.
o Better meat can be obtained in a shorter duration in sheep and fish
 Higher Wool production in Transgenic Sheep:
o Transgenic sheep is made by introducing bacterial genes for synthesis of amino acid
cysteine.
o Such sheep produces higher yield of wool
 Transgenic Pigs to be used for xenotransplantation of human organs:
o Transgenic pigs are produced having human gene which make them more genetically
closer to human beings.
o The organs from such transgenic sheep can be used for transplantation in human with
reduced chances of immunoreactions.

Notes prepared by MRINAL CHOUDHURY Page 9 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

ETHICAL ISSUES

 Ethics: ethics is a set of moral principles and standards by which a community regulates its
behaviour and decides as to the legitimacy and illegitimacy of an activity.

 Bioethics: a set of moral principles and standards which regulate our perceptions as to
exploitation of biological world

 GEAC (Genetic Engineering Approval Committee): Government organisation that takes

decision regarding validity of Genetic Engineering / Genetic Modification research and their
safety of use in the environment.

 Major Bioethical issues are:

o Unfamiliar proteins are introduced into the organisms which react to form toxins and
allergens.
o Gene for high yield introduced into crops can pass into weeds through pollen transfer,
which may increase their population indiscriminately.
o Animals used in biotechnology experiments are made to suffer
o Animals being used to produce biological products are reduced to the status of
factories.
o As a foreign gene is introduced into the organism, it loses the integrity as a species
o The their race to gain supremacy over others, companies and individuals are rushing
for biopatents even of those products which are produced through traditional
knowledge of tribals, communities and societies.
 Two major bioethical issues are: Biopatent and Biopiracy

Biopatent
 Patent: an official right from a government granting an inventor or establishment the sole
monopoly to make and sell a particular article for a certain period.
 Biopatent: an official right from a government granting an inventor or establishment the sole
monopoly to use a particular biological material for commercial exploitation of
i. Strains of microorganisms
ii. Cell lines
iii. Genetically modified plants and animals
iv. DNA sequences
v. Protein encoded by DNA sequences
vi. A particular biotechnological procedure
vii. A production process
viii. Product
ix. Product application
 Ethical issues related to biopatenting: Cases study
o Certain developed countries are issuing broad patents for whole group of organisms
which prevent others from using any related organism or for carrying out any
research.
o Case of Basmati rice:
 27 documented varieties of Basmati rice are grown in India.
 An American company developed a new variety of basmati by crossing an Indian
farmer’s variety and semi dwarf variety.
 The company got patent for the new variety from US patent and Trademark
office, which allowed the company to sell the new variety in the US and abroad.
 The US patent extends to functional equivalents which mean it prevents anyone
else from selling Basmati rice.

Notes prepared by MRINAL CHOUDHURY Page 10 of 11

 Study Notes – Class XII BIOTECHNOLOGY – Applications & Ethical Issues

Biopiracy

 Use of bio-resources by multinational companies and other organisations without proper

authorization from the countries and people concerned without compensatory payments.
 Mostly industrialised nations are involved which are rich financially.
 Underdeveloped nations having rich biodiversity are usually victimized of biopiracy.

Action taken
 Indian Parliament has amended the Indian Patent Bill.
 It now considers emergency granting of patent for research and development initiative.

Notes prepared by MRINAL CHOUDHURY Page 11 of 11