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