➢ Application of Biotechnology includes the following:
o therapeutics, diagnostics,
o genetically modified crops for agriculture,
o processed food, bioremediation,
o waste treatment, and energy production
➢ Critical Research areas of Biotechnology
I.Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme.
II.Creating optimal conditions through engineering for a catalyst to act, and
III.Downstream processing technologies to purify the protein/organic compound.
➢ Application in Agriculture
o Methods for increasing food production:
▪ agro-chemical based agriculture
▪ organic agriculture
▪ genetically engineered crop-based agriculture
o The green revolution succeeded in increasing the food supply but with an increased use of the agrochemicals (fertilizers and pesticides).
o The use of genetically modified crops is a solution to minimize the use of agrochemicals but yet can improve crop production.
o Genetically Modified Organisms (GMO): Any organism (Plants, bacteria, fungi and animals) whose genes have been altered.
o Benefits of GM Plants:
▪ Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
▪ Reduced reliance on chemical pesticides (pest-resistant crops).
▪ Helped to reduce post-harvest losses.
▪ Increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).
▪ Enhanced nutritional value of food, e.g., golden rice, i.e., vitamin ‘a’ enriched rice.
➢ Pest Resistant Plants
➔ Bt Cotton:
o When insects feed on the plants, they also occasionally consume the bacteria on it.
o Many bacterial species such as Bacillus thuringiensis have developed defense mechanisms against insect predation.
o This bacterium produces an intracellular protein crystal that contains a toxic insecticidal protein (cry toxin).
o This insecticidal protein can kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes).
o GM cotton plants based on the gene of Bacillus thuringiensis that produces Bt toxin has been developed.
o Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide resistance to insects without the need for insecticides; in effect creating a biopesticide.
▪ Bt cotton, Bt corn, rice, tomato, potato and soyabean etc.
Mechanism of action of Bt Toxins
o In the Bt Cotton plant when the Bt gene gets expressed, it produces the toxic insecticidal protein.
o When it is inside the bacterium it does not kill the bacteria as it is released as inactive protoxins.
o When it is consumed by the insect, the alkaline condition of the insect gut activates the inactive protoxin by solubilizing the protein crystal to its active toxin form.
o The active toxin binds to the surface of epithelium cells of the midgut and creates pores.
o This causes cell swelling and cell lysis and eventually the insect dies.
Types of Bt genes
o The cry toxins are produced by various cry genes that are effective against specific insect groups.
o The cry toxins released by cryIAc and cryIIAb control the cotton bollworms.
o The cry toxins released by cryIAb controls corn borer.
➔ RNA interference (RNAi):
o It is a method of cellular defense in all eukaryotic organisms.
▪ It eliminates “foreign” RNA molecules—in particular, the double-stranded RNAs produced by many viruses/pathogens.
o This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing).
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| Diagram only for reference |
Making Tobacco Plants Resistant to Nematodes!!
o The nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great reduction in yield.
o In the first step nematode-specific genes were introduced into the tobacco plant.
▪ The vector used was Agrobacterium.
▪ The gene were introduced for both the sense and antisense strand.
o When these genes were expressed in the cells of tobacco plant, it produced both sense and antisense strand RNA.
▪ These two RNA strands are complementary to each other.
▪ This creates a double stranded RNA (dsRNA).
o When there is an actual infection by the nematode, it releases the nematode specific DNA into the host cell that gets expressed as mRNA.
o The dsRNA already present in the host plant (Tobacco) initiates the RNAi mechanism.
▪ This silences the nematode specific RNA by destroying it.
o Thus, this transgenic plant is safe from the nematode as the nematode is unable to survive in this plant.
➢ Application in Medicine
➢ Genetically Engineered Insulin
Importance of Insulin
o Insulin released by the pancreas helps in regulating the blood glucose level in body.
o In case of diabetes mellitus, the body does not produce proper insulin and hence there is an elevated level of blood glucose.
o Such individuals need to take regular dose of insulin to maintain the normal blood glucose level.
Traditional method of obtaining Insulin
o Cattles and pigs were slaughtered and the insulin was extracted from the pancreas of these animals.
o Problems associated with this method:
▪ Allergic reactions in individuals receiving these insulins.
▪ Chance of transfer of disease from the animals to the humans beings.
Structure of Insulin
o In humans the insulin is released an inactive pro-insulin.
o This pro-insulin has three polypeptide chains – A, B and C.
o This inactive insulin undergoes maturation to become active and, in the process, loses the extra C-Polypeptide chain.
o The matured insulin consists of two polypeptide chains A and B linked by disulphide bridges.
Process of production of insulin by rDNA technology
o For the first time an American company Eli Lily (1983) produced the insulin by rDNA technology.
o They prepared two DNA sequences that correspond to the polypeptide chain A and B of human matured insulin.
o These two DNA sequences were then cloned into two separate plasmids of E. coli.
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| Diagram only for reference |
o When these two genes were expressed, in two different host cells, two polypeptide chains (A and B) were formed which was then extracted separately.
o These extracted polypeptide chain A and B were then combined by introducing disulfide bonds artificially to form the matured active insulin.
➢ Gene Therapy
o It is a method that aims to cure an inherited genetic disorder by providing a correct copy of the defective gene to the patient.
o It is a collection of methods that allows correction of a gene defect that has been diagnosed in a child/embryo.
o When the functional gene is inserted to the cell of the individual/ embryo, it is expected to take over the function of the non-functional gene.
First Clinical Gene Therapy (1990): ADA deficiency
o The enzyme adenosine deaminase is essential for the normal functioning of the immune system.
o When the gene responsible for the synthesis of this gene is deleted/mutated the purine metabolism is affected.
Methods to cure ADA deficiency:
o Bone marrow transplantation
o Enzyme replacement therapy
▪ Functional adenosine deaminase enzyme is injected to the patient.
o Gene Therapy
▪ The lymphocytes from the blood of the patient are grown in a culture outside the body.
▪ A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes, which are subsequently returned to the patient.
▪ As these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes.
▪ Permanent Cure:
● If the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages.
➢ Molecular Diagnostics
o Early diagnosis of a disease is crucial towards its treatment and cure.
o The conventional methods of diagnosis (serum and urine analysis, etc) do not provide any early detection for the disease.
o The following methods works towards early diagnosis:
▪ Recombinant DNA technology
▪ Polymerase Chain Reaction (PCR)
▪ Enzyme Linked Immuno-Sorbent Assay (ELISA)
Using PCR for early detection of a bacterial/viral infection.
o When there are symptoms for a particular disease we suspect the presence of the pathogen in the body.
o By then the concentration of the pathogens are generally very high.
o PCR can be used to detect the presence of the pathogen at very low concentration.
▪ This is achieved by amplification of the genetic material (nucleic acid) of the pathogen by PCR.
▪ This enables us to know the presence of the pathogens even if there are no symptoms.
o PCR is now routinely used to detect HIV in suspected AIDS patients.
o It is being used to detect mutations in genes in suspected cancer patients too.
rDNA technology
o A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is used.
o It is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography.
o The clone having the mutated gene will not appear on the photographic film, because the probe will not have complementarity with the mutated gene.
Enzyme Linked Immunosorbent Assay (ELISA)
o It is based on antigen-antibody reaction.
o It can detect the presence of pathogen by detection of either:
▪ the presence of antigens (proteins, glycoproteins, etc.)
▪ the antibodies synthesised against the pathogen.
➢ Transgenic animals
o Animals with modified DNA to possess and express an extra (foreign) gene are known as transgenic animals.
o Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced.
o Following are some reasons to produce transgenic animals:
➔ Study of normal physiology and development:
o Helps in the study of how genes are regulated, and how they affect the normal functions of the body and its development.
o Example-study of complex factors involved in growth such as insulin-like growth factor.
➔ Study of disease:
o Transgenic animals are made to act as models for human diseases.
o These models help in understanding of how genes contribute to the development of diseases.
o Transgenic models exist for many human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s.
➔ Biological Products:
o Biological products that are useful to humans can be produced with the help of transgenic animals.
o This is done by the introduction of the gene that codes for that particular product.
o Examples:
▪ α-1-antitrypsin: used to treat emphysema
▪ treatment of phenylketonuria (PKU) and cystic fibrosis
▪ Rosie – First Transgenic Cow (1997).
● human protein-enriched milk (2.4 grams per liter).
● It contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies than natural cow-milk.
➔ Testing of Vaccine Safety
o Transgenic animals (mice) can be used to test the safety of a new developed vaccine before use in humans.
o Have been used for checking the safety of polio vaccine.
➔ Chemical safety testing
o Here transgenic mice are used for testing the toxicity of drugs.
o Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals.
o They are then exposed to the toxic substances and the effects studied.
o Toxicity testing in such animals will allow us to obtain results in less time.
➢ Ethical Issues
o GEAC (Genetic Engineering Approval Committee):
▪ Regulates the validity of GM research and the safety of introducing GM-organisms for public services in India.
o The modification/usage of living organisms for public services (as food and medicine sources, for example) has also created problems with patents granted for the same.
o Sometimes certain companies are being granted patents for products and technologies that make use of the genetic materials, plants and other biological resources that have long been identified, developed and used by farmers and indigenous people of a specific region/country.
▪ Patents- typically given to an invention by a government authority conferring a right for a set period, especially the sole right to exclude others from making, using, or selling an invention without prior permission from the inventor.
Examples 1: Patent on Basmati Rice
▪ There are an estimated 200,000 varieties of rice in India alone.
▪ Basmati rice is distinct for its unique aroma and flavour and 27 documented varieties of Basmati are grown in India.
▪ There is reference to Basmati in ancient texts, folklore and poetry, as it has been grown for centuries.
▪ In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office.
▪ This allowed the company to sell a ‘new’ variety of Basmati, in the US and abroad.
▪ This ‘new’ variety of Basmati had actually been derived from Indian farmer’s varieties.
▪ Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or a novelty.
▪ The patent extends to functional equivalents, implying that other people selling Basmati rice could be restricted by the patent.
o Example 2: Several attempts have also been made to patent uses, products and processes based on Indian traditional herbal medicines, e.g., turmeric neem.
➢ Biopiracy:
o It refers to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned without compensatory payment.
o Most of the industrialised nations are rich financially but poor in biodiversity and traditional knowledge.
o In contrast the developing and the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources.
o Laws are being developed by some nations to prevent unauthorised exploitation of their bio-resources and traditional knowledge.
o The Indian Parliament has cleared the 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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