Biotechnology and its Applications

Transgenic organisms: • Organisms whose genome has been artificially modified by the introduction of a gene from another organism (foreign/transgene) • Also called genetically modified organisms (GMOs) or recombinant organisms • The foreign gene may be from the same species (cisgenic) or a different species (transgenic) • Created using recombinant DNA technology, Agrobacterium Ti-plasmid, biolistics (gene gun), or other transformation methods Examples: Bt cotton, Bt brinjal, Flavr Savr tomato, Golden Rice, transgenic mice, Rosie (transgenic cow with human alpha-lactalbumin gene in milk) Advantages of transgenic crop plants: 1. Pest resistance (Bt crops): • Bt cotton, Bt brinjal contain Cry genes from Bacillus thuringiensis • Produce Bt toxin (Cry protein) that is toxic to specific insect pests • Reduces insecticide use → less environmental pollution, lower cost • Example: Bt cotton controls bollworm 2. Herbicide tolerance: • Crops engineered to tolerate specific herbicides • Farmers can spray herbicides to kill weeds without harming the crop • Example: Roundup Ready soybean (tolerates glyphosate herbicide) 3. Improved nutritional quality: • Golden Rice: engineered to produce β-carotene (pro-vitamin A) • Addresses vitamin A deficiency in developing countries • Biofortified crops with higher iron, zinc, proteins 4. Disease resistance: • Crops engineered to resist viral, fungal, or bacterial diseases • Example: virus-resistant papaya (ringspot virus resistance) 5. Abiotic stress tolerance: • Drought, salinity, heat, cold tolerance engineered • Important for food security in changing climate 6. Increased yield and shelf life: • Flavr Savr tomato: antisense RNA inhibits polygalacturonase → delayed ripening, longer shelf life 7. Phytoremediation: • Plants engineered to absorb and detoxify heavy metals from contaminated soil

Bt Toxin: • Insecticidal protein (Cry protein, also called delta-endotoxin) produced by the soil bacterium Bacillus thuringiensis (Bt) • Encoded by the cry genes (e.g., cryIAc, cryIIAb for cotton bollworm; cryIAb for maize stem borer) • Bt toxin is a bio-insecticide — natural, target-specific, biodegradable How Bt toxin is produced by Bt bacteria: • During sporulation, Bt bacteria produce protoxin (inactive protein crystals) • Crystals are stored inside bacterial spores Mechanism of action: Step 1 — Ingestion: • Insect larvae ingest Bt bacteria or transgenic plant material containing cry gene Step 2 — Activation: • Insect midgut is alkaline (pH ~9–11) and contains gut proteases • Alkaline proteases convert inactive protoxin → active Bt toxin • Note: In mammals, stomach is acidic (pH ~2) → protoxin NOT activated → safe for mammals Step 3 — Binding: • Active Bt toxin binds to specific receptors on epithelial cells lining the midgut • Receptors are present only in susceptible insects (specific binding = specific toxicity) Step 4 — Pore formation: • Toxin inserts into the cell membrane and forms large pores • Pores disrupt osmotic balance → cells swell and lyse • Midgut epithelium is destroyed Step 5 — Death: • Disruption of midgut → insect cannot feed and digest • Bacteria from soil/spores can invade through disrupted gut • Insect larvae stop feeding within hours and die within 1–3 days Specificity: • Different Cry proteins are toxic to different insects (Lepidoptera, Diptera, Coleoptera) • cryIAc: Tobacco budworm, cotton bollworm • cryIAb: Maize stem borer • cryIVB: Mosquito larvae

Gene Therapy: • The technique of inserting a functional gene into cells of an individual to correct a genetic defect or disease • Treats disease at the genetic level • Somatic gene therapy: gene inserted into somatic (body) cells — not heritable • Germline gene therapy: gene inserted into germ cells — heritable (ethically controversial) Types: • Ex vivo: Cells removed from patient → gene introduced in lab → cells returned to patient • In vivo: Gene delivered directly into the patient's cells Vectors used: Retroviruses (integrate into genome), Adenoviruses, Adeno-associated viruses, Liposomes Gene therapy for ADA Deficiency (Adenosine Deaminase Deficiency): ADA deficiency: • Caused by mutation in the ADA gene on chromosome 20 • ADA enzyme (adenosine deaminase) is essential for immune system function • Without ADA → accumulation of toxic metabolites (deoxyadenosine triphosphate, dATP) • Toxic to lymphocytes (T and B cells) → immune deficiency (SCID — Severe Combined Immunodeficiency) • Affected children cannot fight infections; live in sterile environments (bubble babies) Gene therapy procedure: 1. Lymphocytes from patient's blood are removed 2. Functional ADA cDNA introduced into lymphocytes using retroviral vector 3. Transformed lymphocytes with functional ADA gene are grown in culture 4. Genetically modified cells introduced back into patient (ex vivo gene therapy) 5. Modified lymphocytes produce functional ADA enzyme → immune system partially restored Limitation: • Lymphocytes have a limited life span; procedure must be repeated • Bone marrow gene therapy (stem cells) — longer-lasting solution • Currently patients can also be treated with PEG-ADA (modified ADA enzyme) First successful gene therapy: Anderson et al., 1990 — a 4-year-old girl with ADA-SCID treated with gene therapy.

Ethical issues in GMOs (Genetically Modified Organisms): 1. Environmental concerns: • Gene flow / cross-pollination: GM crops may transfer transgenes to wild relatives → 'superweeds' resistant to herbicides or pests • Biodiversity loss: GM monocultures may replace diverse traditional crop varieties • Harm to non-target organisms: Bt toxin may affect beneficial insects (e.g., monarch butterfly larvae initially thought to be affected by Bt corn pollen) • Unforeseen ecological consequences of introducing new genes into ecosystems 2. Food safety concerns: • Allergenicity: Transgenes might produce novel proteins that cause allergic reactions • Horizontal gene transfer: Antibiotic resistance marker genes in GM crops could theoretically transfer to gut bacteria • Long-term health effects: Insufficient long-term safety data in humans • Consumer right to know: Mandatory labelling of GM food debated globally 3. Socioeconomic and ethical concerns: • Corporate control: Seed patents held by large corporations (Monsanto, now Bayer) → farmers forced to buy seeds annually (terminator technology) • Biopiracy: Using genetic resources from developing countries without consent or compensation • Equity: Technology mostly benefits wealthy farmers; poor farmers may be marginalised • Playing God: Ethical objection to humans altering genomes of organisms 4. Animal welfare in transgenic research: • Transgenic animals used in research may suffer from inserted gene effects • Cloning and genetic modification of animals raises welfare concerns 5. Human gene therapy ethics: • Somatic gene therapy (to cure disease): Generally accepted • Germline modification: Alters all future generations; permanent, heritable changes — widely condemned (example: He Jiankui edited human embryos in 2018 — condemned globally) • Enhancement vs therapy: Gene editing for enhancement (intelligence, height) — strongly opposed 6. Regulatory frameworks: • Each country has different regulations; lack of international consensus • India: GEAC (Genetic Engineering Appraisal Committee) regulates GM crop approvals • Precautionary principle: Should GMOs be released until proven safe?

Key achievements of the Human Genome Project (HGP), completed 2003: 1. Complete sequencing: • Sequenced approximately 3.2 billion base pairs (bp) of the human genome • Covered ~92% of genome initially; near-complete sequence now available (T2T genome, 2022) 2. Gene catalogue: • Identified ~20,000–25,000 protein-coding genes (far fewer than expected — scientists expected ~100,000) • Annotated all genes with their locations and functions (ongoing) 3. Non-coding DNA discovery: • Only ~1.5% of human DNA codes for proteins • Large proportion of genome is non-coding: regulatory elements, repetitive sequences, pseudogenes, transposons • Led to ENCODE project (Encyclopedia of DNA Elements) to characterise functional non-coding DNA 4. Comparative genomics: • Humans share 99.9% DNA with each other (individual variation is tiny) • 98–99% similarity with chimpanzee genome • Evolutionary relationships clarified across species 5. SNP mapping: • Identified millions of Single Nucleotide Polymorphisms (SNPs) • Used for population genetics, disease association studies, pharmacogenomics 6. Medical applications: • Identification of disease genes (BRCA1/2, CFTR, LRRK2, etc.) • Basis for personalised medicine, pharmacogenomics • Development of genetic tests for predisposition to diseases 7. Technological revolution: • Drove development of next-generation sequencing (NGS) — reduced cost from $3 billion to under $500 • Bioinformatics as a major discipline 8. Ethical, Legal, Social Implications (ELSI): • 5% of HGP budget dedicated to ELSI research — genetic privacy, discrimination, patenting of genes

ELISA (Enzyme-Linked Immunosorbent Assay): • A laboratory technique that uses antibodies and enzyme-linked colour reactions to detect and quantify specific proteins (antigens or antibodies) in a sample • Highly sensitive, specific, and widely used diagnostic tool Principle: • Based on antigen-antibody binding • One antibody captures the target; a second enzyme-linked antibody detects it • Enzyme (e.g., HRP — Horseradish Peroxidase, or Alkaline Phosphatase) converts a colourless substrate into a coloured product • Colour intensity is proportional to the amount of antigen/antibody in the sample Types: 1. Direct ELISA: Antigen coated on plate; enzyme-labelled antibody detects it 2. Indirect ELISA: Antigen on plate; unlabelled primary antibody + enzyme-labelled secondary antibody 3. Sandwich ELISA: Capture antibody on plate; antigen added; enzyme-labelled detection antibody (most sensitive) 4. Competitive ELISA: Unlabelled antigen in sample competes with labelled antigen for antibody Applications: 1. Diagnosis of infections: • HIV diagnosis: ELISA detects anti-HIV antibodies (initial screening test) • Hepatitis B, Hepatitis C detection • Dengue, malaria, COVID-19 detection (antigen ELISA) 2. Allergy testing: • Detects IgE antibodies specific to allergens 3. Autoimmune disease diagnosis: • Detects autoantibodies (e.g., anti-dsDNA for lupus, anti-CCP for rheumatoid arthritis) 4. Pregnancy testing: • Detects hCG (human chorionic gonadotropin) in urine or blood 5. Food safety: • Detection of food allergens, pathogens, toxins in food samples 6. Drug monitoring: • Measures drug levels in blood 7. Research: • Quantification of cytokines, hormones, proteins in research samples