Biotechnology and its Applications

🎯 Key Points

• Bt toxin: Bacillus thuringiensis Cry proteins → protoxin (inactive) → activated by alkaline gut pH of insect → kills larvae; cry1Ac, cry2Ab in Bt cotton (bollworm); cry1Ab in Bt corn (stem borer)
• RNAi: dsRNA triggers RISC to degrade complementary mRNA → gene silencing; used in tobacco against nematode Meloidogyne incognita
• Golden Rice: β-carotene (provitamin A) pathway genes from daffodil/maize inserted → addresses vitamin A deficiency
• Human insulin: A and B chains produced separately in E. coli then combined → Humulin (Eli Lilly, 1982, first GM pharma product)
• Gene therapy: ADA deficiency - T-lymphocytes extracted, functional ADA gene inserted via retroviral vector, re-infused; not permanent (must repeat)
• Molecular diagnostics: PCR (amplify tiny pathogen DNA/RNA); ELISA (antigen-antibody based - HIV, hepatitis); Southern blotting
• Transgenic animals: Rosie the cow (human α-lactalbumin in milk); transgenic sheep for blood clotting Factor IX (haemophilia)
• Biopiracy: exploitation of bio-resources/knowledge without sharing benefits; e.g., Basmati rice patent (USA), turmeric healing (USA) - both challenged by India

GM Crops & Pest Resistance

• Bt cotton: cry1Ac + cry2Ab genes; resistant to bollworm (Helicoverpa); first GM crop approved in India
• Bt corn: cry1Ab; protects against corn borer
• Herbicide-tolerant plants: bar gene from Streptomyces hygroscopicus → resistance to herbicide bialaphos
• Flavr Savr tomato: antisense RNA blocks polygalacturonase → delayed ripening (first GM food, USA 1994)

Biopharmaceuticals

• Insulin: pre-proinsulin gene (minus signal sequence) in E. coli → A + B chains produced separately + joined by disulfide bonds
• Human growth hormone (HGH), erythropoietin (EPO), Factor VIII all produced in GM organisms
• Streptokinase (clot buster): produced by Streptomyces but now via GM bacteria

Gene Therapy

• ADA deficiency (autosomal recessive): ADA enzyme needed for lymphocyte survival; patients immunodeficient
• Somatic gene therapy: functional gene delivered into patient's own cells; not heritable
• Germ-line gene therapy: changes in egg/sperm/early embryo; heritable - ethically controversial

Molecular Diagnostics & Bioethics

• PCR: even one copy of pathogen DNA/RNA detectable; used for HIV, TB, cancer mutations (early cancer)
• ELISA: antigen detected by enzyme-linked antibody → colour change proportional to concentration; used for HIV screening
• Biosafety guidelines (EPA, GEAC in India): mandatory before release of GM organisms
• GEAC (Genetic Engineering Appraisal Committee): approves field trials and release of GMOs in India
• Biopiracy: Convention on Biological Diversity (CBD) - countries must share benefits of bio-resources

Pest-Resistant Plants: RNA Interference (RNAi)

• RNA interference (RNAi) is a method of cellular defence and gene silencing in which a specific mRNA is prevented from being translated by a complementary double-stranded RNA (dsRNA) molecule
• The tobacco plant is infected by the nematode Meloidogyne incognita, which reduces yield; nematode-specific genes were introduced into the host plant using Agrobacterium vectors
• The introduced DNA produces both sense and antisense RNA that pair to form a dsRNA, which initiates RNAi and silences the specific nematode mRNA
• As a result the parasite cannot survive in the transgenic host, so the plant is protected from the pest

Transgenic Animals

• Transgenic animals have had their DNA manipulated to carry and express an extra (foreign) gene; over 95% are mice, but rats, rabbits, pigs, sheep, cows and fish have also been produced
• Normal physiology and development: used to study how genes are regulated and how they affect body functions (e.g. study of insulin-like growth factor)
• Study of disease: many serve as models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's, helping test new treatments
• Biological products (pharming): valuable proteins can be produced in animal milk; e.g. the transgenic cow Rosie produced milk enriched with human alpha-lactalbumin
• Vaccine and chemical safety testing: transgenic animals are used to test the safety of vaccines and the toxicity of chemicals

Ethical Issues, Biopiracy and Patents

• Genetic modification of organisms can have unpredictable effects when they are released into ecosystems, so a regulatory body is required to evaluate biosafety before use
• GEAC (Genetic Engineering Appraisal Committee): the Indian body that examines the validity of GM research and the safety of introducing GM organisms for public services
• Biopiracy: the use of bioresources and traditional knowledge by companies or organisations without proper authorisation from, or compensation to, the countries and people concerned
• Cases challenged by India include patents (later revoked) on Basmati rice, the wound-healing property of turmeric (haldi), and uses of neem
• India has amended its Patents Act and enacted laws to prevent unauthorised exploitation of its bioresources and traditional knowledge

Benefits of Genetically Modified (GM) Crops

• Made crops more tolerant to abiotic stresses such as cold, drought, salt and heat
• Reduced reliance on chemical pesticides (as in pest-resistant Bt crops)
• Helped to reduce post-harvest losses of produce
• Increased efficiency of mineral usage by plants, preventing early exhaustion of soil fertility
• Enhanced the nutritional value of food, for example Golden rice (vitamin A enriched)

Golden Rice and Biofortification

• Golden rice is a genetically engineered variety of rice that is rich in provitamin A (beta-carotene), which the body converts into vitamin A
• It was developed to combat vitamin A deficiency, a major cause of blindness in developing countries where rice is the staple food
• Genes for beta-carotene synthesis were introduced into the rice grain (endosperm), giving it a characteristic golden-yellow colour
• Biofortification is the breeding or engineering of crops for higher vitamin, mineral, protein or healthier-fat content to improve public nutrition

Production of Recombinant Human Insulin

• Insulin is made of two short polypeptide chains, chain A and chain B, joined together by disulphide bridges
• In the human body, insulin is first synthesised as a longer inactive precursor called proinsulin, which carries an extra stretch called the C-peptide
• The C-peptide is removed during maturation to form the functional insulin; this C-peptide is not present in the mature hormone
• In 1983, the company Eli Lilly prepared the two DNA sequences corresponding to the A and B chains, introduced them into E. coli, and produced the chains separately
• The chains A and B were then extracted and combined by creating disulphide bonds to form human insulin, marketed as Humulin

Biopiracy and Patents: Neem, Turmeric and Basmati

• Biopiracy is the use of bio-resources and traditional knowledge by multinational companies or organisations without proper authorisation or compensation to the country/people concerned
• Patents were granted (and later challenged) on the wound-healing properties of turmeric and the insecticidal/medicinal properties of neem, both long known to Indian traditional knowledge
• A US company also obtained patents relating to Basmati rice, a variety traditionally grown in India
• To prevent such unauthorised exploitation, the Indian Parliament has amended patent laws, and the country protects its biodiversity and indigenous knowledge under the Biological Diversity Act
• In India, the GEAC (Genetic Engineering Appraisal Committee) makes decisions on the validity and safety of GM research and the release of GM organisms for public use