Biotechnology and Its Applications Notes

Biotechnology Applications

Overview of Biotechnology

  • Deals with industrial-scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants, and animals.

  • Applications include: therapeutics, diagnostics, GM crops, processed food, bioremediation, waste treatment, and energy production.

  • Three critical research areas:

    • Improved organism (microbe/enzyme) as a catalyst.

    • Optimal conditions for catalyst action through engineering.

    • Downstream processing to purify products.

Biotechnological Applications in Agriculture

Options for Increasing Food Production

  1. Agrochemical-based agriculture

  2. Organic agriculture

  3. Genetically engineered crop-based agriculture

Limitations of Green Revolution

  • The Green Revolution successfully increased food supply but was insufficient for the growing population.

  • Increased yields were due to improved crop varieties, management practices, and agrochemicals.

  • Agrochemicals are expensive for developing-world farmers.

  • Conventional breeding methods have limitations in speed and efficiency.

Tissue Culture

  • Whole plants can be regenerated from explants (any plant part) under sterile conditions.

  • Totipotency: The capacity to generate a whole plant from any cell/explant.

  • Nutrient medium requirements: carbon source (sucrose), inorganic salts, vitamins, amino acids, and growth regulators (auxins, cytokinins).

  • Micro-propagation: Producing thousands of plants through tissue culture.

  • Somaclones: Genetically identical plants produced via micropropagation.

  • Applications: production of food plants (tomato, banana, apple) on a commercial scale.

  • Recovery of healthy plants from diseased plants (using meristem culture).

Protoplast Fusion

  • Isolated protoplasts (cells without cell walls) from different plant varieties can be fused.

  • Somatic hybrids: New plants formed from hybrid protoplasts.

  • Somatic hybridization: The process of fusing protoplasts.

  • Example: Pomato (tomato + potato hybrid), which lacked desired commercial characteristics.

Genetically Modified Organisms (GMOs)

  • Plants, bacteria, fungi, and animals with altered genes.

  • Benefits:

    • Increased tolerance to abiotic stresses (cold, drought, salt, heat).

    • Reduced reliance on chemical pesticides (pest-resistant crops).

    • Reduced post-harvest losses.

    • Increased efficiency of mineral usage.

    • Enhanced nutritional value (e.g., golden rice).

    • Production of alternative resources (starches, fuels, pharmaceuticals).

Pest-Resistant Plants

  • Bt toxin: Produced by Bacillus thuringiensis.

  • Bt toxin gene cloned and expressed in plants to resist insects, reducing insecticide use.

  • Examples: Bt cotton, Bt corn, rice, tomato, potato, and soyabean.

Bt Cotton

  • Bacillus thuringiensis strains produce proteins toxic to insects (lepidopterans, coleopterans, dipterans).

  • Protein crystals containing insecticidal protein are formed during bacterial growth.

  • The Bt toxin exists as inactive protoxins within the bacterium.

  • Activation of toxin: Ingestion by insect → alkaline pH of gut solubilizes crystals → toxin activated.

  • Mechanism: Toxin binds to midgut epithelial cells, creates pores, causes cell swelling/lysis, and insect death.

  • Specific Bt toxin genes (e.g., cryIAc, cryIIAb) are isolated and incorporated into crop plants.

  • cryIAc and cryIIAb control cotton bollworms; cryIAb controls corn borer.

RNA Interference (RNAi) for Pest Resistance

  • Nematode Meloidegyne incognitia infects tobacco roots and reduces yield.

  • RNAi is used as a cellular defense mechanism in eukaryotic organisms.

  • Mechanism: dsRNA binds to and silences specific mRNA, preventing translation.

  • Source of dsRNA: viral infections or mobile genetic elements.

  • Process:

    • Nematode-specific genes are introduced into the host plant using Agrobacterium vectors.

    • Sense and antisense RNA are produced, forming dsRNA.

    • RNAi silences nematode mRNA.

    • The parasite cannot survive, and the plant is protected.

Biotechnological Applications in Medicine

Recombinant Therapeutics

  • Recombinant DNA technology enables mass production of safe and effective therapeutic drugs.

  • Recombinant therapeutics do not induce unwanted immunological responses.

  • Approximately 30 recombinant therapeutics are approved worldwide; 12 are marketed in India.

Genetically Engineered Insulin

  • Management of adult-onset diabetes is possible with regular insulin intake.

  • Earlier insulin was extracted from slaughtered cattle and pigs, causing allergic reactions in some patients.

  • Human insulin can now be produced by bacteria.

  • Insulin consists of two polypeptide chains (A and B) linked by disulfide bridges.

  • In humans, insulin is synthesized as a pro-hormone containing an extra C peptide, which is removed during maturation.

  • Challenge: Assembling insulin into mature form using rDNA techniques.

  • In 1983, Eli Lilly produced two DNA sequences corresponding to chains A and B of human insulin in E. coli.

  • Chains A and B were produced separately, extracted, and combined by creating disulfide bonds.

Gene Therapy

  • Corrective therapy for hereditary diseases.

  • Genes are inserted into a person’s cells/tissues to treat disease.

  • Genetic defects are corrected by delivering a normal gene to compensate for the non-functional gene.

  • First clinical gene therapy: 1990, for a 4-year-old girl with adenosine deaminase (ADA) deficiency.

  • ADA deficiency: caused by deletion of the gene for adenosine deaminase, which is crucial for immune function.

  • Treatments: bone marrow transplantation or enzyme replacement therapy (functional ADA injections).

  • Limitations: These treatments are not completely curative.

  • Process:

    • Lymphocytes from the patient's blood are grown in culture.

    • A functional ADA cDNA (using a retroviral vector) is introduced into these lymphocytes.

    • Lymphocytes are returned to the patient, requiring periodic infusions.

    • Introducing the ADA gene into marrow cells at early embryonic stages could be a permanent cure.

Molecular Diagnosis

  • Early diagnosis and understanding pathophysiology are essential for effective treatment.

  • Recombinant DNA technology, Polymerase Chain Reaction (PCR), and Enzyme Linked Immuno-sorbent Assay (ELISA) are used for early diagnosis.

  • PCR can detect very low concentrations of bacteria or viruses by amplifying their nucleic acid.

  • Applications: HIV detection, detecting mutations in cancer patients, identifying genetic disorders.

  • Method: Single-stranded DNA or RNA (probe) tagged with a radioactive molecule is hybridized to complementary DNA in a clone of cells, followed by autoradiography.

  • ELISA: Based on antigen-antibody interaction.

  • Detection: Identifying antigens (proteins, glycoproteins) or antibodies synthesized against the pathogen.

Transgenic Animals

  • Animals with manipulated DNA to possess and express an extra (foreign) gene.

  • Examples: rats, rabbits, pigs, sheep, cows, and fish; 95% are mice.

  • Reasons for production:

    • Normal physiology and development: Study gene regulation and effects on normal body functions (e.g., insulin-like growth factor).

    • Study of disease: Models for human diseases (cancer, cystic fibrosis, rheumatoid arthritis, Alzheimer’s).

    • Biological products: Production of medicines, such as human protein (α-1-antitrypsin) to treat emphysema. First transgenic cow, Rosie, produced human protein-enriched milk.

    • Vaccine safety: Testing vaccine safety before human use (e.g., polio vaccine).

    • Chemical safety testing: Toxicity/safety testing using animals more sensitive to toxic substances.

Ethical Issues

  • Regulation is needed for the manipulation of living organisms.

  • Ethical standards are required to evaluate the morality of human activities affecting living organisms.

  • Genetic modification of organisms can have unpredictable ecological results.

  • GEAC: Genetic Engineering Approval Committee (Indian Government organization) makes decisions on the validity of GM research and the safety of GM organisms.

  • Patents on modified organisms and genetic materials create problems.

  • Public anger against companies patenting products and technologies using genetic materials developed by farmers and indigenous people.

Biopiracy

  • Use of bio-resources by multinational companies without proper authorization or compensatory payment.

  • Industrialized nations: financially rich but poor in biodiversity and traditional knowledge.

  • Developing/underdeveloped world: rich in biodiversity and traditional knowledge.

  • Exploitation of traditional knowledge can save time and effort in commercialization.

  • Growing realization of injustice in benefit sharing.

  • Nations are developing laws to prevent unauthorized exploitation.

  • The Indian Parliament has addressed these issues in amendments to the Indian Patents Bill.

Basmati Rice Example

  • Basmati rice has a unique aroma and flavor, with 27 documented varieties in India.

  • In 1997, an American company patented Basmati rice, allowing them to sell a ‘new’ variety derived from Indian farmers' varieties.

  • The patent extends to functional equivalents, restricting other sellers.

Traditional Herbal Medicines

  • Attempts have been made to patent uses, products, and processes based on traditional Indian herbal medicines (e.g., turmeric, neem).

  • Vigilance is needed to counter these patent applications to protect our legacy.