ZOOLOGY

BIOTECHNOLOGY
&
IT’S APPLICATIONS

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 BIOLOGY
AIIMS 2020
Class : XIII (All) BIOTECHNOLOGY & IT’S APPLICATIONS NOTES
BIOTECHNOLOGY & IT’S APPLICATIONS

 Green biotechnology – Agriculture

 Red biotechnology – Medicine
 White / Grey biotechnology – Industrial products
 Blue biotechnology – Marine ecosystem

 Areas of research in biotechnology

1. Providing the best catalyst in form of Improved organisms, usually a microbe or pure enzyme
2. Creating optimal conditions through engineering for a catalyst to act
3. Downstream processing technologies to purify protein / organic compound

 Biotechnological applications in agriculture

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Three options for increasing food production are–
1. Agro – chemical based agriculture(fertilizers, pesticides etc.)
2. Organic agriculture/farming( biofertilizer etc)
3. Genetically Engineered Crop – Based agriculture

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 Green revolution (mid 1960s)– food supply tripled but yet not enough for growing population
 Father of green revolution– Norman Ernest Borlaug(USA)


1.
Green revolution Resulted from –

Use of improved crop varieties (High yielding)

N
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2. Better management practices(irrigation, machine use, soil conservation etc.)
3. Use of agro–chemicals (fertilizers/pesticides)

 Hindrances–
O

1. Agro chemicals too expensive

2. Harmful effect on environment (increase soil salinity & decrease soil fertility)
3. Increased yield not possible using conventional breeding
O

 Overcoming of above problems by

 GMO (Genetically Modified organism)
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 Genetic modification has –

1. Made crops more tolerant to abiotic stress (cold, draught, flood, storm, salt, acids, heat)
2. Reduced reliance on chemical pesticides (pest resistant crop)
3. Reduced post harvest losses
4. Increased efficiency of mineral usage by plant (prevents early exhaustion of soil fertility)
5. Enhanced nutritional value of food, eg. vitamin A enriched rice

 The GM crops have been used to create Tailor–made plants to supply alternative resources to the industries in form
of Starches, fuels & pharmaceuticals (like hirudin)

 Production of Hirudin protein (anticoagulant) from transgenic Brassica napus seeds

1. Isolation of hirudin gene from common cow leech (Hirudinaria granulosa)/chemically synthesized
2. Insertion of hirudin gene into genome of Brassica napus(rate of seed production fast) – A.tumifaciens
3. Plant tissue culture
4. Expression of hirudin gene into Hirudin protein in all plants

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5. Isolation of Hirudin protein from seeds of these plants
6. Purification & storage for sale (intramuscular inj. Incase of internal blood clots)

 Desired gene – hirudin gene

 Donor – cow leech
 Vector –A. tumifaciens
 Transgenic plant / GMP – Brassica napus plant

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 Golden rice / Vitamin –A rich rice –

 Developed by Ingo Potrykus and Peter Beyer

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 Transgenic variety of rice (Oryza sativa), contains  – carotene (provitamin A–inactive state of vitamin A)
 Vector – A. tumifaciens
 Donor – carrot / daffodil



Grain – yellow colored

Production of Protein rich Pulses

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 Production of Bt–cotton : Insecticidal
O
O
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Cotton ball: (a) destroyed by ballworms; (b) a fully mature cotton ball

 Ball worms destroy the crop

1. Isolation of cry gene from Bacillus thuringiensis

2. Insertion of this gene into genome of cotton plant
3. Plant tissue culture
4. Expression of cry gene into Cry protein in all plants
5. Plants become insect resistant (ballworm)

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Bt toxin :–

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 Bt toxin – endotoxin in bacterium Bacillus thuringiensis (soil bacterium, inactive precursor form)
 Bt toxin gene has been cloned from bacteria and expressed in plants to provide resistance to insects, without need
for insecticide (bio–pesticides)
 Pest resistant plants – decrease use of pesticide


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Some strains of B. thuringiensis produce proteins that kill certain insect (lepidopterans–ballworms/tobacco
budworm/armyworm, coleopterans– beetles, dipterans–flies, mosquitoes)
 Forms protein crystals during a particular phase of growth (sporulation), which contains toxic insecticidal protein
 Cry protein – crystallizable protein

 This toxin does not kill bacillus

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 because it exists as inactive protoxin
 but once an insect ingest this, it is converted into active form due to alkaline pH of gut which solubilise crystals
O

 Desired gene – Bt gene/cry gene

 Donor – Bacillus thuringiensis
 Vector –A. tumifaciens

O

Transgenic plant / GMP – cotton plant

 Bt cotton(introduced in India in 2002), Bt corn, Bt rice, Bt tomato, Bt potato, Bt brinjal, Bt soyabean etc
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Bt – toxin :

 Choice of gene – depends upon crop and the targeted pests (these toxins are insect – group specific)
 Genes cryIAc and cryIIAb – control cotton ballworms
 cryIAb – controls corn stem borer

 Note :

 Examples of transgenic organisms which are obtained by direct expression of wanted / desired gene

 RNAi / RNA interference / anti–sense RNA / m–RNA silencing :

 Discovered by Fire & Mello (1998)– Nobel prize in 2006

 Takes place in all eukaryotic organisms as method of cellular defense

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 ds–RNA ( sense & anti–sense RNA)

Dicer (Ribonuclease enzyme)
siRNA (21–25 Nucleotide long small interfering RNA)

inactive RISC (RNA Induced Silencing Complex)

active RISC

bind with m–RNA

Breaking of m–RNA by RISC enzymes

m–RNA SILENCED

No translation / no expression


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No protein formation

 RNAi involves silencing of a specific m–RNA due to complementary ds–RNA molecule that binds to and prevents
translation of m–RNA (Silencing)

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 Source of ds–RNA :

1. Infection by virus with RNA genome

2.
3.
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Transposons / mobile gene elements which replicate via RNA intermediate
Using A.tumifaciens –––––introduce gene into host cell–––––produce ds – RNA
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 Pest / Nematode Resistant Plant by RNAi :

In Nematode –
O
O
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 Meloidegyne incognitia – called root-knot nematode
 Nematode resistant plant obtained by blocking the expression of normal functional gene

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 It is Due to complimentary ds– RNA which binds & prevent translation of m–RNA
 Source of this ds–RNA can be from an infection by a virus (having RNA genome ) or mobile genetic element
(=Transposon), which replicate via an RNA intermediate
 Using Agrobacterium vector, nematode specific genes introduced into host plant
 introduction of DNA was such that it produced both sense & anti–sense RNA in host cells

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 These 2 RNA, being complimentary to each other forms a ds–RNA that initiated RNAi
 Thus silenced m–RNA of nematode
 The parasite could not survive in the transgenic plant(host), expressing a specific interfering RNA



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Transgenic plant becomes protected from parasite (PEST RESISTANT PLANT)

Flavr Savr Tomatoes/ Mac Gregor tomatoes – by Calgene company (1994)

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 Prevent Post harvest Losses/Delayed Fruit Ripening/Increased shelf life
 Enzyme responsible for ripening of tomatoes – "Pectinase" (Gene X : PG/ Polygalactorunase)
 1st commercially grown genetically engineered food, available in market
O
O
ET

 Note :

 Flavr – Savr tomato – transgenic plant which is obtained by blocking the expression of normal functional gene
(Antisense – mRNA technology)
 Size of tomato increases 6–8 times
 Its flavor turned sour

 Biotechnology application in medicine

 Advantages–

1. Mass production
2. Safe and more effective drugs
3. Do not induce unwanted immunological responses as from non human resources

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1. Genetically Engineered Insulin
2. Gene therapy
3. Monoclonal antibodies
4. Vaccines

 About 30 recombinant therapeutics approved world over

 In India, 12 hour marketed

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Protein Used in the Treatment
Erythropoietin Anemia
Blood Clotting Factor VIII Haemophilia – A
Follicle stimulating hormone Reproductive disorders
Granulocyte colony stimulating factor(GCSF) & GMCSF Cancer

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Human Insulin Diabetes mellitus
Interferon–2 & 2 Leukemia(CML), other cancers
Interferon– Cancer, rheumatoid arthritis
Human Interleukins
Human GH /Somatotrophin
Hepatitis B vaccine
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Cancer, immune disorders
Growth disorders In children
Hepatitis B
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Streptokinase (thrombolytic) Acute myocardial infarction
Tissue plasminogen activator
 Genetically Engineered Insulin:
O

 Earlier extracted from pancreas of slaughtered cattles and pigs (developed allergy /reactions to foreign proteins)
 Banting & Best – 1st extracted insulin from pancreas of dog (1921)
 Sanger – 1st studied / gave molecular structure ( aa sequence)

O

insulin gene – 10 Kb(on chromosome 11)

 Insulin – two short polypeptide chains (A–21 aa /63 nucleotides & B–30 aa / 90 nucleotides) linked together by
disulphide bridges
 Synthesized as pro–hormone in mammals, contains extra C peptide (31 aa /93 nucleotides, links A & B chain, not
ET

present in mature insulin, removed during maturation)

Maturation of pro–insulin into insulin (simplified)

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 Main challenge – getting insulin assembled into mature form
 In 1983, Eli Lilly (America company) prepared 2 DNA sequences corresponding to A and B chains and introduced
them in plasmids of E.coli separately to produce insulin chains, extracted and combined both chains by creating
disulphide bonds
 Produced 1st genetically engineered insulin / hormone (5th July 1983)
 Humulin (trade name)–
 regular insulin
 Short acting insulin
 Takes about 30 min to reach blood stream
 Works about 3–6 hours

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


Gene of interest – insulin gene
Vector – plasmid (insert size upto 15 Kb)
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Fig. 12.11. Steps involved in gene transler for the production of human insulin.
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Host – E.coli (Transgenic)
 World Diabetes Day – 14th November ( Banting's birthday)
 Humolog –
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 Rapid / fast acting Insulin ( gets initiated faster in 10–15 min)

 Last 2–4 hours
 Eli lily & Ranbaxy introduced ' insulin analogue'
O

 Insulin cant be taken orally because it is degraded in alimentary canal

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 Why does eukaryotic gene not get expressed in prokaryote?

 As Prokaryotes lack intron (transcription coupled to translation ), hence intron mediated transcription ( Splicing,
Ligase absent)
 Thus, cDNA (obtained from m–RNA by reverse transcriptase enzyme, contains only exons, no introns)is used to
clone eukaryotic gene in prokaryote

 Gene therapy –

 Methods to correct gene defect diagnosed in a child/embryo, genes are inserted into persons cells and tissues to
treat a disease
 Replacement of non–functional gene with a normal gene

 Types of gene therapy –

A) Basis = complexity of process

1. In–vivo : gene correction done inside body of patient

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2. Ex–vivo : cell / organ is taken out from body of patient, gene correction done inside lab & corrected cell / organ again
re–inserted to patient's body

B) Basis = type of cell given gene therapy

1. Somatic cell gene therapy –

 Gene therapy of somatic cells, Not inharitable

 Successful in Cystic fibrosis, SCID

2. Germ line gene therapy –

 Gene therapy of sex cells / germ cells

 Not successful yet
 By injecting microinjection in germ cell / embryo, may cause harmful mutations
 If successful, many inheritable gene mutations will be treated

 First clinical gene therapy :

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 Given in 1990 (14th sept)
 To four year girl Ashanthi D'Silva
 with adenosine deaminase (ADA) deficiency (essential for immune system functioning)
 By Dr. William Anderson

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 At National Institute of Health, USA

 SCID (Severe Combined Immuno Deficiency) – ADA:



Deletion of ADA gene (25% cases).
This gene codes for enzyme adenosine deaminase
N
SI
 Involved in purine metabolism, irreversibly deaminates adenosine, converting it to related nucleoside inosine––––
deribosylated by purine nucleoside phosphorylase enzyme–––– converted to hypoxanthine)
O

 Deoxyadenosine is toxic to lymphocytes (Essential for immune function)

 ADA– SCID : Bubble babies’
O

 Treatment modalities (ADA–SCID) –

1. Enzyme replacement therapy (ERT – IV injection of ADA) – functional ADA Enzyme IV

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2. Bone Marrow Transplantation – BMT /Haemopoeitic Stem Cell Transplantation–HSCT

 both not completely curable

3. Gene therapy –
a) lymphocytes from blood of patient grown in culture outside body
b) functional ADA cDNA (using retroviral vector) is introduced into these lymphocytes (host)
c) Engineered Lymphocytes are subsequently returned to patient

 Limitation : As these cells are not immortal, patient requires periodic infusion of genetically engineered lymphocytes
(up to 3 weeks)
 Permanent cure if gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages
 Desired gene –ADA gene
 Insert –ADA cDNA
 Vector / helper – Retrovirus
 Host – patient's lymphocyte

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Trick for Gene Therapy

 Monoclonal antibody / Magic bullets –

 Highly specific against a particular antigen ( only one kind of antibody)

 Called ' Hybridoma Technology'
 Developed by Kohler & Milstein (1975), Nobel prize in 1984

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Fig. 25.6 Hybridoma Technique and production of monoclonal antibodies

1.
2.
3.
Myeloma cells – cancerous cells
Hybridoma cell –
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Antigen stimulated B– Lymphocyte from spleen –Antibody producing
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 Antibody producing trait of B–Lymphocyte
 Rapid division trait of Myeloma cell
4. Culture medium – HAT
( Hypoxanthine – Aminopterine(toxic to non hybrid cells) – Thymidine)
O

Trick for Monoclonal Antibody –

O
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 Use of Monoclonal Antibody –

1. Diagnosis of disease
2. Treatment of disease
3. Prevent rejection in transplantation as Immuno–suppressant . Eg– OKT–3

 Molecular diagnosis :

1. Conventional method of diagnosis (blood/serum/urine/stool analysis etc)–early detection not possible

 Presence of pathogens normally suspected – when disease symptom produced (pathogen concentration already
very high in body)

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2. Modern methods ( rDNA technology/ PCR/ ELISA/ Blotting) – early diagnosis
A) PCR :

 Very low concentration of pathogen can be detected by amplification of their nucleic acid by PCR (when symptom not
yet visible)
 Routinely used to detect HIV in suspected AIDS patient
 Used to detect mutations in genes in suspected cancer patient's
 Technique to identify many genetic disorder

 Application of PCR –

1. Dx of pathogens
2. Dx of specific mutation – phenylketonuria, muscular dystrophy
3. Dx of specific microorganisms – HIV, TB, Sickel cell anemia etc.
4. Dx of Plant pathogens – viroids, CMV etc
5. DNA finger printing
6. Palaeontology – Fossil study
7. Prenatal diagnosis – avoidance of abortion

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B) Autoradiography (rDT) :

 Method allowing detection / localization of radioactive isotope with a biological sample

 Single stranded RNA/DNA, tagged with radioactive molecule like P32/ fluorescent marker (probe) is allowed to

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hybridized to its complementary DNA in a clone of cells.
 The clone having mutated gene will not appear on photographic film, because probe will not have complementarity
with mutated gene

C) ELISA :
(Enzyme Linked Immuno–Sorbent Assay)
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SI
 Principle –Antigen–Antibody interaction
 Detection of Infection by pathogen –
 by presence of antigens (proteins, glycoproteins etc.) or by detecting anti– bodies synthesized against pathogen
O

 Types of ELISA :
O

1. Direct ELISA – test for antigen presence

2. Indirect ELISA– test for antibody presence
3. Sandwich ELISA– test for antigen presence
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4. Competitive ELISA

The most commonly used enzyme include Peroxidase and Alkaline phosphatase

 Direct ELISA steps –

1. Blood of suspect taken out
2. Blood centrifuged
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3. Serum isolated
4. Serum added to a 96 well microtitre plate (coated with HIV antigen & chromogenic substrate)
5. Known Enzyme linked Ab (Primary Ab conjugate)
6. Rinse thoroughly
7. Addition of chromogenic substrate for enzyme
 If positive for HIV, blue color produced – HIV infection
 If negative for HIV, no color produced – No HIV Infection

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 Indirect ELISA steps :

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1. Blood of suspect taken out
2. Blood centrifuged
3.
4.
5.
Serum isolated N
First Antigen is adsorbed / attaches on wall of microtitre plate
Serum, suspected of containing antibody (Primary antibody) is added
SI
6. Rinse thoroughly
7. Enzyme linked Ab (secondary antibody conjugate), capable of reacting with constant region of other Ab or to Ag–
Ab complex is added
8. Rinse thoroughly
O

9. Addition of chromogenic substrate for enzyme

 Indirect ELISA commonly used for HIV detection

O

 Screening test for HIV - ELISA

 Confirmatory test for IV-Western blotting
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 If positive for HIV , blue color produced – HIV infection

 If negative for HIV , no color produced – No HIV Infection

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 Sandwich ELISA steps :

1. Blood of suspect taken out

2. Blood centrifuged
3. Serum isolated
4. First Antibody (primary Ab) is adsorbed / attaches on wall of microtitre plate
5. Serum, suspected of containing antigen is added
6. Rinse thoroughly
7. Enzyme linked Ab (Secondary Ab conjugate), reacting with Ag-Ab complex is added
8. Rinse thoroughly
9. Addition of chromogenic substrate for enzyme

 If positive for HIV, blue color produced – HIV infection

 If negative for HIV, no color produced – No HIV Infection
 Development of color on addition of chromogenic substrate indicates presence of antigens/ antibodies
 Intensity of color – indication for titre

 Transgenic /Genetically Modified Organism (GMO) –

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 The one which contains a gene from any other organism / They have their DNA manipulated to possess and express
an extra- foreign gene
 GMPs, GMAs, GMMs

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 Transgene / foreign / alien gene –

 a gene from other organism

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Genetically Modified Organisms and their applications
(commonly cloned organisms)
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GM Crop Application
1. Bt cotton Pest resistant, herbicide tolerant, highly resistant to ballworm infestation
2. Golden rice Vitamin- A rich rice
O

3. potatoes High protein content

4. Corn, brinjal Insect resistant
5. Banana, tomato Edible vaccines
O

6. Soyabean, maize Herbicide resistant

7. Flavr savr tomato Increased shelf life, delayed ripening, better nutritional quality
8. Brassica napus Hirudin production
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9. Tobacco plant Nematode(pest) resistant

GM Animal Application
1. cattle Production of therapeutic proteins in milk (nurtitious & safe for human infants)
2. Pigs organ transplantation(rejection risk minimum)
3. Fish High protein content (twice the size by HGH gene insertion)
4. Super mouse contains breast cancer causing human gene, helps researchers to study disease
5. Monkey ANDi gene for green fluorescent protein (GFP)
6. Cow Rosie -gene for human alpha-lactalbumin protein
7. sheep more wool production, treatment of emphysema
8. goats spider silk production

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 Microbe Application
1. Bacillus thuringiensis (bacterium) Production of Bt toxin (highly safe, potent & biodegradable insecticide for plant
protection)
2. Escherichia coli (gut bacteria) Production of human insulin, growth hormone, interferon, interleukins, FSH,
erythropoeitin etc.
3. Pseudomonas fluoroscence (bacteria) Prevention of frost damage to plants on which it grows (strawberries)
4. Pseudomonas putida/ Scavenging of oil spills by digesting hydrocarbons of crude oil (Bioremediation-
super bug (bacteria) use of microorganism metabolism to remove pollutants)
5. Rhizobium meliloti (bacteria) Nitrogen fixation by ‘nif’ gene incorporation in cereal crops
6. Klebsiella (bacteria) ‘nif’gene incorporation
7. Agrobacteiu m tumifaciens Disease (tumor) resistant crops
(bacteria)
8. Trichoderma (fungus) Production of enzyme chitinase for biocontrol of fungal diseases in plants
9. Trametes (fungus) Removal of lignin from wood pulp (increased paper production)

 TRANSGENIC ANIMALS :

 They have their DNA manipulated to possess and express an extra- foreign gene

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 95% are mice (rats, rabbits, pigs, sheep, cows, fish)

 Advantages :

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1. Normal physiology and development eg. Study of complex factors involved in growth like insulin – like growth factor
2. Study of disease – models exists for cancer, cystic fibrosis, rheumatoid arthritis, alzheimer’s
3. Vaccine safety – Transgenic mice to test safety of polio vaccine (could replace use of monkeys, if successful)
4. Chemical safety testing /toxicity testing

4. Biological products
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SI
a) Human protein (a-1-antitrypsin) to treat emphysema (1st transgenic sheep ‘Tracy’)
b) Treatment of phenylketonuria (PKU) and Cystic fibrosis
c) In 1997, first transgenic cow, Rosie – produce human protein enriched milk (2.4gm/litre), contained human alpha –
O

lactalbumin (nutritionally more balance product for human babies than natural cow milk)
d) Larger sheep for more wool production
e) Goats – spider silk producing gene
O

 CLONING :


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Production of organisms with exactly similar genetic make up.

 Types of cloning :
1. Gene cloning (via PCR & use of vectors)
2. Microbe cloning
3. Plant cloning / plant tissue culture
4. Animal cloning

 Why Microbe cloning is more advantageous ?

1. Rate of reproduction is higher

2. Culturing of organism is very easy & economic (cheap)

 On lab scale – on pertidishes using Agarose medium

 On industrial scale – using bioreactors / fermentors

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 2 principles of Cloning :

1. Cellular totipotency :
 If a cell is propogated on culture medium & gives rise to complete organism
 Followed by all plant cells & embryonic stem cells of animals
 Discovered by Steward etal by taking 2 mg phloem cells of carrot

2. Pleuriopotency :
 If a cell is propogated on culture medium & gives rise to single organ or part of an organ
 Shown by all animal cells, except embryonic stem cells

 Animal cloning :

 Involves both principles of pleuriopotency & cellular totipotency

 Very tedious & skill oriented process

 Ian Wilmat et.al at Roseline – Franklin Research Institute, Scotland cloned calf of wild boar, ‘Frosty’ (first mammalian
clone but unsuccessful) but died just after birth

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 First successful mammalian clone- Dolly sheep (cloned by adult somatic cell)

 Created by Ian Wilmat et.al

 At Roseline – Franklin Research Institute, Scotland

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 On 5th July 1996
 Died on 14th February 2003 due to lung disease & arthritis

Dolly, the sheep : N

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O
O
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 First amphibian clone - Toad = Xenopus laevis

 Created by Gurdon et.al of Germany

 Donor – intestinal cell of female frog – I
 Host – unfertilized egg cell of female frog – II
 Surrogate mother not required as in amphibia, fertilization is external

 First cloned monkey –ANDi (green fluoroscent protein gene inserted)

 Cloning of Asian Gaur (Bos gauras) - A revolution because

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 Saved species from extinction
 Two different species were used

 Polly & Molly (1997) –

 transgenic sheep cloned

 Ethical issues :

 Bioethics - set of standard to regulate our activities in relation to biological world

 GMOs can have unpredictable results when introduced into ecosystem

 GEAC (Genetic Engineering Approval Committee) by Indian government – make decision regarding validity of GM
research, safety of introducing GMO for public services

 Biopatents – rights granted to a person / company for its discovery of a biological product

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 Novelty, Non-obviousnes and Utility
 Biopiracy - use of Bio- resources by multinational companies and other organizations without proper authorization
from the countries and people concerned without compensatory payment
 Industrilized nations – Rich financially, poor in biodiversity and traditional knowledge (developing and under-

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developed world – vise a versa)

 EXAMPLES OF BIOPIRACY :
1.
2.
Biopatency of Brassica family by USA N
Biopatency of basmati rice by US Company- In 1997, an American company got patent on basmati (Indian basmati
was crossed with semi- dwarf varieties)
SI
 Basmati rice – unique aroma & flavour
 2 lac varieties of rice in India alone (27 varieties of basmati)
3. Biopatency of Turmeric (wound healing property) - by American company
4. Biopatency of Neem (fungicidal property) – by American company
O

5. Biopatency of Pentadiplandra brazzeana of S.Africa – by American company

 produce Brazzein protein, 2000 times sweeter than normal cane sugar & don’t interfere with blood glucose levels
O

 If not vigilant, others may encash our rich legacy

 Indian parliament recently cleared second amendment of the Indian patents bill, that takes such issues into
consideration, including patent terms emergency provisions and research and development initiative
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 Bioweapon & Biowar/Bioterrorism :

 War or mass destruction by use of living organisms, esp. microbes (bacteria, virus, fungus & their toxins as
Bioweapons)
 Economical
 Invisible
 Examples of Biowar –
1. Use of Agent Orange during World War-II
 Powerful herbicide, used by US military forces against Vietnam to clear forest
2. Spread of Anthrax in Germany (Early 2000s)
 Bioweapon agent – spores of bacteria Bacillus anthracis
 Inert carrier – Talcom powder
 Delivery system – Envelopes
3. Yersinia pestis – plague
4. Clostridium botulinum – in frozen food, paralysis of skeletal system
5. Alpha virus / Brain virus

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