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The critical areas of biotechnology�

Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme.
Creating optimal condition through engineering for a catalyst to act.
Downstream processing technologies to purify the protein/organic compound.
BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE:
Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).
Advantages of Genetic Modification in plants.

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Bt Cotton�

Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans(tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes).
B.thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxicinsecticidal protein.
These proteins are present in inactive protoxin form, but become active toxin in the alkaline pH of insect gut.
The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of insect
Specific Bt toxin genes were isolated form B. thuringiensis and genetically transferred to several plants such as cotton.
Crystal proteins are produced by a gene called cry in B. thuringiensis.
The protein coded by genes cryIAc and cryIIAb control the cotton bollworms.
The protein coded by gene cryIAb controls corn borer.

Pest resistant plants

Several nematodes parasitize a wide variety of plants and animals including human beings.
A nematode Meloidegyne incognitia infects the root of tobacco plants and causes a great reduction in yield.
Strategy based on RNA interference (RNAi) prevents this infestation.
Process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of mRNA

Steps of RNA interference

Double stranded RNA is produced endogenously or exogenously.
Using Agrobacterium vectors nematode specific genes were introduced into the host plant (tobacco plant).
Introduction of DNA produces both sense and antisense RNA in the host.
These two RNA’s being complementary to each other formed a double stranded (dsRNA) that initiated RNAi.
The dsRNA injected into the host plant from outside called exogenous dsRNA.
The dsRNAs are cleaved into 21-23 nt segments (“small interfering RNAs”, or siRNAs) by an enzyme called Dicer.
siRNAs are incorporated into RNA-induced silencing complex (RISC)
Guided by base complementarity of the siRNA, the RISC targets mRNA for degradation.
The consequence was that the parasite could not survive in a transgenic host.

BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

Biotechnology enables mass production of safe and more effective therapeutic drugs.
Recombinant therapeutics does not induce unwanted immunological responses as is common in case of similar products isolated from non-human sources.
At present around 30 recombinant therapeutics, approved for human-use.

Genetically Engineered Insulin

Taking insulin at regular interval of time is required for adult-onset diabetes.
Previously the source of insulin was the slaughtered cattle and pigs.
This insulin caused allergy in some patients.
Each insulin made of two short polypeptide chains; chain A and chain B that are linked together by disulphide linkage.
Insulin synthesized in pancreas as pro-hormone which is a single polypeptide with an extra stretch called C-peptide.
C-peptide is removed during matured insulin.
In 1983 Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E.coli to produce insulin chains.
Chain A and chain B produced separately, extracted and combined by creating disulfide bonds to form mature human insulin.

Gene therapy�

Gene therapy is an attempt to cure hereditary or genetic diseases.
Genes are inserted into a person’s cells and tissue to treat the disease.
The first clinical gene therapy was given in 1990 to a 4-yr old girl with adenosine deaminase (ADA) deficiency.
This enzyme is required for breakdown of deoxyadenosine into uric acids.
In the absence of ADA toxic deoxyadenosine is accumulated and destroy the infection fighting immune cells called T-cells and B-cells.
This disorder is caused due to the deletion of the gene for adenosine deaminase in chromosome

Treatment:

Treated by bone marrow transplantation.
Enzyme replacement therapy, involving repeated injections of the ADA enzyme
Lymphocytes from the blood of the patient are grown in a culture. A functional ADA cDNA is then introduced into these lymphocytes and returned into the body.
The patient required periodic infusion of genetically engineered lymphocytes because these cells are not immortal.
Functional ADA cDNA introduced into cells at early embryonic stages, could be the permanent cure.

Molecular diagnosis�

Early detection of disease is not possible by conventional methods (serum and urine analysis)
Molecular diagnosis techniques:
Recombinant DNA technology.
Polymerase chain reaction (PCR)
Enzyme linked Immuno-sorbent Assay (ELISA)
Very low concentration of a bacteria or virus can be amplified and detected by PCR.
It used to detect genetic disorders.
PCR is use full to mutation in genes in suspected cancerous patient:

A single stranded DNA or RNA tagged with radioactive molecule (probe) is allowed to hybridize to its complementary DNA in a clone of cells followed by detection using autoradiography.
The clone having mutated gene unable make complementary bonding of probe, hence not appears in photographic film.

TRANSGENIC ANIMALS

Animals that have an alien DNA which able to express in it is called transgenic animals.
Reasons for creation of transgenic animals:
Normal physiology and development:

Study of disease:

Transgenic animals are designed to understand how genes contribute to the development of disease like cancers, cystic fibrosis, rheumatoid arthritis and Alzheimer’s.

Biological products:

α-1-antitrypsin used to treat emphysema.
Proteins for treatment for PKU and cystic fibrosis.
Transgenic cow Rosie, produce human protein enriched milk (2.4 gm/lit. human α-lactalbumin)

Vaccine safety:

Transgenic mice are being developed and use in testing the safety of vaccines before they are used for humans.
Polio vaccine is tested in mice.

Chemical safety testing

ETHICAL ISSUES�

GEAC (Genetic Engineering Approval Committee) set up by Indian Govt, which will make decisions regarding validity of GM research and safety of introducing GM-organisms for public services.
A patent is the right granted by a government to an inventor to prevent others from commercial use of his invention.
Patents granted for biological entities and for products derived from them; these patents are calledbiopatents.
27 documented varieties of Basmati are grown in India.
Biopiracy is the term used to refer to the use/exploit or patent, of biological resources by multinational companies and other organizations without proper authorization from the countries and people concerned without compensatory payment.