12. BIOTECHNOLOGY AND ITS APPLICATIONS Biotechnology has many applications such as biopharmaceuticals, therapeutics, diagnostics, genetically modified crops, processed food, bioremediation, waste treatment and energy production. Biotechnology has 3 critical research areas: a. Providing the best catalyst in the form of improved organism usually a microbe or enzyme. b. Creating optimal conditions through engineering for a catalyst to act. c. Downstream processing technologies to purify the protein/organic compound. APPLICATIONS IN AGRICULTURE 3 options for increasing food production: a. Agro-chemical based agriculture: It uses fertilizers & pesticides. Expensive. Causes environmental pollution. b. Organic agriculture: Expensive. c. Genetically engineered crop-based agriculture: It uses genetically modified crops. Genetically Modified Organisms (GMO) are the plants, bacteria, fungi & animals whose genes are altered by manipulation. Advantages of genetic modification in plants: It makes crops more tolerant to abiotic stresses (cold, drought, salt, heat etc.). Pest-resistant crops reduce the use of chemical pesticides. - Itreduces post-harvest losses. It increases efficiency of mineral usage by plants (it prevents early exhaustion of soil fertility).~~ Tt enhances nutritional value of food. E.g. Golden rice (Vitamin A enriched rice). To create tailor-made plants to supply alternative resources (starches, fuels, pharmaceuticals etc.) to industries. Pest Resistant Plants They act as bio-pesticide. It reduces the need for insecticides. E.g. Bt cotton, Bt corn, rice, tomato, potato, soyabean etc. Bt Cotton: - Some strains of Bacillus thuringiensis have proteins that kill insects like coleopterans (beetles), lepidopterans (tobacco budworm, armyworm) & dipterans (flies, mosquitoes). - B. thuringiensis forms an insecticidal protein (Bt toxin) crystal during a phase of their growth. It does not kill the Bacillus as it exists as inactive protoxins. - When an insect ingests the toxin, it becomes active due to alkaline pH of the gut which solubilise the crystals. Toxin binds to surface of mid-gut epithelial cells creating pores. It causes cell swelling and lysis and death of the insect. - Bt toxin genes were isolated from B. thuringiensis and incorporated into crop plants such as cotton. - Most Bt toxins are insect-group specific. They are coded by ery genes. E.g. proteins encoded by crylAc & cryIIAb genes control cotton bollworms. Protein of cryIAb gene controls corn borer. Nematode resistance in tobacco plants: - Anematode Meloidogyne incognita infects the roots of tobacco plants causing a reduction ingrowth, It does not kill the Bacillus as it exists as inactive protoxins. - When an insect ingests the toxin, it becomes active due to alkaline pH of the gut which solubilise the crystals. Toxin binds to surface of mid-gut epithelial cells creating pores. It causes cell swelling and lysis and death of the insect. - Bt toxin genes were isolated from B. thuringiensis and incorporated into crop plants such as cotton. - Most Bt toxins are insect-group specific. They are coded by cry genes. E.g. proteins encoded by cryIAc & cryIIAb genes control cotton bollworms. Protein of cryIAb gene controls corn borer. Nematode resistance in tobacco plants: - Anematode Meloidogyne incognita infects the roots of tobacco plants causing a reduction in yield. - It can be prevented by RNA interference (RNAi) strategy. - RNAi is a method of cellular defense in all eukaryotic organisms. It prevents translation of a specific mRNA (silencing) due to a complementary dsRNA molecule. - The source of this complementary RNA is from an infection by RNA viruses or mobile genetic elements (transposons) that replicate via an RNA intermediate. Isolate Nematode-specific genes (DNA). It is introduced into host plant using Agrobacterium vectors. It produces both sense & anti-sense RNA in host cells. These RNAs are complementary. So they form double stranded (ds) RNA. It initiates RNAi and silences the specific mRNA of nematode. Thus the parasite cannot survive in a transgenic host expressing specific interfering RNA.APPLICATIONS IN MEDICINE - Recombinant DNA technology helps for mass production of safe and more effective therapeutic drugs. - Products from non-human sources cause unwanted immunological responses. But recombinant therapeutics does not have such problems. : At’ present, about 30 recombinant therapeutics have been approved. Of these, 12 are being marketed in India. LG : Engi . - Insulin is used to manage adult-onset diabetes. - Insulin from the pancreas of animals (cattle & pigs) causes allergy or other types of reactions to the foreign protein. Now, it is possible to produce human insulin using bacteria. Insulin consists of two short polypeptide chains (chain A & chain B) that are linked by disulphide bridges. - In mammals, insulin is synthesized as a pro- hormone (pro-insulin). It is processed to become mature and functional hormone. S & srrne Insulin * — Bpeptide ~ Free C peptide - - The pro-hormone contains an extra stretch called C peptide. This is removed during maturation into insulin.- The pro-hormone contains an extra stretch called C peptide. This is removed during maturation into insulin. - In 1983, Eli Lilly (an American company) prepared two DNA sequences corresponding to A & B chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A & B were combined by creating disulfide bonds to form human insulin (Humulin). 2. Gene Therapy - It is a method to correct a gene defect in a child/embryo. - Here, genes are inserted into a person’s cells and tissues to treat a hereditary disease. It compensates for the non-functional gene. ~ First clinical gene therapy (1990) was given to a 4-year old girl with adenosine deaminase (ADA) deficiency. ~ This is caused due to the deletion of a gene of adenosine deaminase (an enzyme for the functioning of immune system). It can be cured by bone marrow transplantation or by enzyme replacement therapy (injection of ADA). But these are not completely curative. - Gene therapy for ADA deficiency: Collect lymphocytes from the patient’s blood and grow in a culture — Introduce a functional ADA cDNA into lymphocytes (using a retroviral vector) . They are returned to the patient.3M Di 5 = Conventional methods (serum & urine analysis) are not suitable for early diagnosis of diseases. It is possible by techniques such as Recombinant DNA technology, PCR & ELISA. PCR (Polymerase Chain Reaction): - Presence of a pathogen is normally suspected only based on symptoms. By this time, the concentration of pathogen is already very high in the body. - However, very low concentration of a bacteria or virus can be detected by amplification of their nucleic acid by PCR. ~ Uses of PCR: o To detect HIV in suspected patients. o To detect gene mutations in suspected cancer patients. o To identify many other genetic disorders. - A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is hybridized to its complementary DNA in a clone of cells. It is detected by autoradiography. The clone having mutated gene will not appear on photographic film, because the probe will not have complementarity with mutated gene. ELISA (Enzyme Linked Immuno-Sorbent Assay): - Itis based on antigen-antibody interaction. - Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesized against the pathogen.TRANSGENIC ANIMALS - These are the animals whose genome has been altered by introduction of a foreign gene by manipulation. - Eg. Transgenic rats, rabbits, pigs, sheep, cows and fish. - Over 95% of the transgenic animals are mice. Benefits of transgenic animals ' To study regulation of genes and their action on normal physiology & development: E.g. Study of insulin-like growth factor. Genes (from other species) that alter formation of this factor are introduced and the biological effects are studied, This gives information about biological role of the factor. - To study the contribution of genes in the development of a disease and thereby new treatments: E.g. transgenic models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis & Alzheimer’s. : Biological products: Some medicines contain expensive biological products. Transgenic animals can be used to produce biological products by introducing genes which codes for a particular product. They are used to treat diseases such as emphysema, phenylketonuria (PKU), cystic fibrosis etc, E.g. human protein (a-1- antitrypsin) used to treat emphysema. In 1997, Rosie (first transgenic cow) produced human protein-enriched milk (2.4 gm per litre). It contains human a-lactalbumin. It is nutritionally more balanced product for human babies than natural cow-milk.