Topics to be Learn :
  • Biotechnology
  • Principles and Processes of Biotechnology
  • Methodology for r-DNA technology
  • Applications of Biotechnology
  • Bioethics
  • Effects of Biotechnology on the Environment
  • Effects of Biotechnology on Human Health
  • Biopatent and Biopiracy
Biotechnology

  • Term first used by Karl Ereky in 1919 to describe large-scale pig production.
  • Defined as: ‘Development and utilization of biological forms, products, or processes for maximum benefits to humans and other life forms’.
  • OECD Definition (1981): Application of scientific and engineering principles to process materials using biological agents for goods and services aimed at human welfare.

Scientific and Engineering Principles:

  • Include: Microbiology, Genetics, Biochemistry, Chemical Engineering, Mathematics, Statistics, Computers, and Industrial Processes.
  • Biological Agents: Plants, Animal cells, Microorganisms, Enzymes, or their products.

Development of Biotechnology:

Traditional Biotechnology:

  • Based on fermentation technology using microorganisms.
  • Examples: Preparation of curd, ghee, soma, vinegar, yogurt, cheese, wine.

Modern Biotechnology:

  • Use of r-DNA technology, PCR, microarrays, cell culture.
  • Ownership of technology and its socio-political impact.

Principles and Processes of Modern Biotechnology:

Based on two core techniques:
(i) Genetic Engineering:
  • Manipulation of genetic material to achieve desired outcomes using in vitro processes.
  • Definition (Smith): ‘Formation of new combinations of heritable material by insertion of nucleic acid molecules into host organisms for replication and propagation’.
  • Also known as recombinant DNA technology or gene cloning.
(ii) Chemical Engineering:
  • Focuses on maintaining sterile environments for producing products like vaccines, antibodies, enzymes, organic acids, vitamins, therapeutics.

Technique of Gene Cloning and rDNA Technology:

  • Involves transferring a gene of known function into a cell that doesn’t naturally contain it, using a suitable vector.
  • The transferred gene is replicated and passed to future progeny.

Tools and Techniques for Gene Cloning/rDNA Technology

  • Techniques in r-DNA Technology (Based on molecular weight): Gel permeation, Osmotic pressure, Ion exchange chromatography, Spectroscopy, Mass spectrometry, Electrophoresis

Electrophoresis:

  • Used for separating charged molecules like DNA, RNA, and proteins by applying an electric field.
  • Types of Electrophoresis: Agarose gel electrophoresis, PAGE (Polyacrylamide Gel Electrophoresis), SDA PAGE (Sodium Dodecyl Sulfate PAGE)

Polymerase Chain Reaction (PCR):

  • Used for in vitro gene cloning to produce billions of copies of a desired DNA/RNA segment with high accuracy and specificity.
Basic Requirements of PCR:
  • DNA containing the desired segment to be amplified.
  • Excess of forward and reverse primers: Synthetic oligonucleotides of 17-30 nucleotides, complementary to DNA sequences.
  • dNTPs (four types): dATP, dGTP, dTTP, dCTR.
  • Thermostable DNA polymerase (e.g., Taq DNA polymerase) that can withstand high temperatures (90-98°C).
  • Mg++ ions in appropriate quantities.
  • Thermal cycler: A device to carry out PCR reactions.

Three Essential Steps in PCR:

1. Denaturation:
  • Reaction mixture is heated to 90-98°C.
  • Hydrogen bonds in DNA break, resulting in the separation of two DNA strands.
2. Annealing of Primer:
  • Reaction mixture is cooled to 40-60°C.
  • Primers pair with their complementary sequences in the single-stranded DNA (ssDNA).
3. Extension of Primer:
  • Temperature is increased to 70-75°C.
  • Thermostable Taq DNA polymerase adds nucleotides to the 3' end of the primer using ssDNA as a template.
  • Duration: About 2 minutes.
In an automatic thermal cycler, these three steps repeat 20-30 times.
After ‘n’ cycles, 2^n copies of the DNA segment are synthesized.

Biological Tools for Gene Cloning/r-DNA Technology

(1) Enzymes:

  • Examples: Lysozymes, Nucleases (exonucleases, endonucleases, restriction endonucleases), DNA ligases, DNA polymerases, alkaline phosphatases, reverse transcriptases, etc.
  • Nucleases: Cut the phosphodiester bonds of polynucleotide chains.
  • Types of Nucleases: Exonucleases: Cut nucleotides from the ends of DNA strands. Endonucleases: Cut DNA from within.

Restriction Endonucleases (Restriction Enzymes):

  • Cut DNA at specific recognition sites (4 to 8 nucleotides long), often with internal symmetry.
  • Can produce fragments with: Blunt ends, Cohesive/sticky ends, Staggered ends (short, single-stranded projections)
Example:
  • Enzyme EcoRI recognizes and cuts the following sequence:
    3' ----C T T A A G ----5'
    5' ----G A A T T C ----3'
  • Palindrome: Reads the same in the opposite direction on both strands.
Types of Restriction Enzymes:
  • Type I: Function as both endonuclease and methylase (e.g., EcoKI).
  • Type II: Have separate cleaving and methylation activities (e.g., EcoRI, BgII).
  • Type III: Cut DNA at specific non-palindromic sequences (e.g., HpaI, MboII).

(2) Cloning Vectors (Vehicle DNA):

  • Vectors: DNA molecules that carry foreign DNA and replicate within the host cell.

Examples of Vectors:

  • Plasmids (e.g., T1 plasmid of Agrobacterium tumefaciens, pBR 322, pUC)
  • Bacteriophages (e.g., M13, lambda virus)
  • Cosmid, Phagemids, BAC (Bacterial Artificial Chromosome), YAC (Yeast Artificial Chromosome), Transposons, Baculoviruses, and MACS (Mammalian Artificial Chromosomes).

Plasmids as Cloning Vectors:

  • Small, extra-chromosomal, double-stranded circular DNA forms that replicate autonomously.
  • Found in bacterial cells, yeast, and animal cells.
  • Known as replicons due to their autonomous replication in suitable hosts.
  • Commonly used in r-DNA technology due to their ability to replicate in E. coli.
  • Effective plasmid vectors have:Replication origin, Marker gene for antibiotic resistance, Control elements (e.g., promoter, operator, ribosome binding site), A region for foreign DNA insertion.
  • Constructed plasmids: pBR 322, pBR320, paCYC177.
T1 plasmid of Agrobacterium tumefaciens is a key vector for DNA transfer in plants:
  • Contains T DNA, a transposon that integrates into plant chromosomes.
  • Helps create transgenic plants through DNA insertion.

(3) Competent Host:

  • Bacteria used for cloning include Bacillus haemophilus, Helicobacter pyroli, and E. coli.
  • E. coli is commonly used for transformation with recombinant DNA.

Methodology for rDNA Technology: Steps Involved in Gene CloningThe steps involved in gene cloning:
  • Isolation of DNA (gene) from the donor organism.
  • Insertion of the desired foreign gene into a cloning vector (vehicle DNA).
  • Transfer of r-DNA into a suitable competent host or cloning organism.
  • Selection of the transformed host cell.
  • Multiplication of the transformed host cell.
  • Expression of the gene to obtain the desired product
(i) Isolation of DNA (Gene) from the Donor Organism:
  • Obtain the desired gene from the donor organism.
  • Cells of the donor organism are: Sheared with a blender, Treated with a detergent to isolate genetic material, DNA is then isolated and purified using various techniques, Purified DNA can be spooled onto a glass rod.
  • DNA is cleaved using restriction endonucleases.
  • Select the restriction fragment containing the desired gene for cloning (foreign DNA or passenger DNA).
  • Alternatively, genes can be sourced directly from a genomic library or cDNA library.
(ii) Insertion of Desired Foreign Gene into a Cloning Vector (Vehicle DNA):
  • Foreign DNA is inserted into a cloning vector like bacterial plasmids or bacteriophages (e.g., lambda phage, M13).
  • Commonly used plasmid: pBR 322.
  • Plasmids are: Isolated from bacteria. Cleaved using the same restriction enzyme (RE) used for the desired gene.
  • DNA ligase joins the foreign DNA to the plasmid DNA.
  • Resulting DNA is known as recombinant DNA (r-DNA) or chimeric DNA.
(iii) Transfer of r-DNA into a Suitable Competent Host or Cloning Organism:
  • r-DNA is introduced into a competent host cell (often a bacterium).
  • Host cell takes up naked r-DNA via transformation and integrates it into its own DNA, expressing the trait controlled by the passenger DNA.
  • Divalent Ca++ ions assist the transfer of r-DNA.
  • Common Cloning Organisms: E. coli and Agrobacterium tumefaciens.
  • Competent host cells that have taken up r-DNA are termed transformed cells.
  • Methods to introduce DNA include electroporation, microinjection, lipofection, shotgun, ultrasonification, biolistic method, etc.
  • In plant biotechnology, transformation often uses Ti plasmids of A. tumefaciens.
(iv) Selection of the Transformed Host Cell:
  • Use marker genes on the plasmid vector to isolate recombinant cells from non-recombinant cells.
  • Example: pBR322 vector with ampicillin-resistant and tetracycline-resistant genes. Using PstI RE removes the ampicillin resistance gene, making recombinant cells sensitive to ampicillin.
(v) Multiplication of Transformed Host Cell:
  • Transformed host cells are cultured in fresh media.
  • Host cells divide and multiply.
  • Recombinant DNA also multiplies within the cells.
(vi) Expression of Gene to Obtain Desired Product:
  • Desired products like enzymes or antibiotics are produced.
  • Products are then separated and purified through downstream processing using bioreactors.

Proteins Produced by r-DNA Technology and Their Applications:

Applications of Biotechnology

Biotechnology spans a wide range of scientific applications across various sectors like healthcare, agriculture, industry, environment, and genomics.

(1) Healthcare Biotechnology:
  • Provides targeted and personalized therapeutic and diagnostic solutions, including: Organ transplant, Stem cell technology, Genetic counselling, Forensic medicine, Gene probes, genetic fingerprinting, and karyotyping.
  • Production of human insulin using r-DNA technology.

Vaccines:

  • A vaccine is a biological preparation that provides active acquired immunity against a specific disease.
  • Made from weakened or killed forms of microorganisms, toxins, or surface protein antigens.

Vaccine Production:

  • Types of vaccines: Recombinant vaccines, Naked DNA vaccines, Viral vector vaccines,Plant-derived vaccines
  • Modern diagnostic test kits include rickettsial, bacterial, and viral vaccines with radio-labelled biological therapeutics for imaging and analysis.

Oral Vaccines:

Production of Edible Vaccine:

  • Engineered edible plant parts produce an immunogenic protein.
  • When consumed, the protein is recognized by the immune system.
  • Gene encoding for the immunogenic protein is isolated and inserted into a suitable vector.
  • Recombinant vector is transferred into the plant genome.
  • Gene expression in plant results in the synthesis of immunogenic proteins.
  • Consumption of these plant parts vaccinates against specific pathogens.

Production of 'Melt-in-the-Mouth' Vaccines:

  • Administered by placing under the tongue, allowing direct entry into the bloodstream.
  • Example: Flu vaccine produced by Bacillus.
  • Benefits include comfort of administration, low cost, and easy storage.

Advantages of Edible Vaccines:

  • Engineered edible plant parts produce an immunogenic protein.
  • Active when administered orally.
  • Offer low cost, ease of administration, and simple storage.
  • Effective in vaccinating humans and animals against various pathogens.

(2) Agriculture

Application of Biotechnology in Agriculture: Focuses on the development of genetically modified organisms (GMOs), including:

  • Bt Cotton: Provides resistance to pests and improves agricultural productivity.
  • Pest-resistant plants: Reduces the need for chemical pesticides.
  • Improved crop yields: Enhances nutritional quality and resilience to environmental conditions.

Applications of Tissue Culture:

  • Micropropagation: Enables large-scale propagation of plants in a short duration.
  • Storage of Germplasm: Maintains clones of plants with recalcitrant seeds or highly variable seeds. Recalcitrant seeds: Sensitive to dehydration and freezing, leading to challenges in storage and viability maintenance.

Know This:

  • Recalcitrant Seeds: These seeds suffer from a reduction in moisture content below certain levels. Freezing can cause subcellular damage, leading to loss of viability.

(3) Gene Therapy

Definition:

  • A technique used to treat genetic disorders by replacing, altering, or supplementing a defective or missing gene.
Delivery methods include:
  • Ex vivo delivery: Gene is introduced outside the body, then cells are returned to the patient.
  • In vivo delivery: Direct introduction of therapeutic genes into target cells within the patient.
  • Use of Virosomes (Liposome + inactivated HIV) and bionic chips for gene delivery.

Delivery Methods:

1. Ex Vivo Delivery:
  • Cells are removed from the patient, modified outside the body, and reintroduced.
  • Example: Parkinson’s disease, a neurological disorder.
2. In Vivo Delivery:
  • Therapeutic genes are directly introduced into target tissues.
  • Example: Injecting genes directly into tumors to treat cancer.
3. Virosomes and Bionic Chips: These methods use advanced vectors to deliver genes into the cells.

Applications of Gene Therapy:

  • Replace missing or defective genes.
  • Deliver genes to promote the destruction of cancer cells.
  • Induce cancer cells to revert to normal cells.
  • Vaccinate by delivering bacterial or viral genes.
  • Stimulate the immune response through DNA delivery.
  • Inhibit viral replication with therapeutic genes.
  • Promote or inhibit the growth of new tissue.
  • Stimulate the healing of damaged tissues.

Forms of Gene Therapy:

(i) Germ Line Gene Therapy:

  • Modifies germ cells (e.g., sperms, eggs, or early embryos).
  • The genetic correction is passed on to future generations.
  • Highly effective in genetic disorder treatment, but not preferred in humans due to ethical and technical concerns.

(ii) Somatic Cell Gene Therapy:

  • Modifies somatic cells (e.g., bone marrow, liver, endothelial cells).
  • Changes affect only the treated individual, not passed to offspring.
  • Considered the only viable option for clinical trials in humans.
  • Applied in treating conditions such as: Cancer, Rheumatoid arthritis, Severe Combined Immunodeficiency (SCID), Gaucher’s disease, Familial hypercholesterolemia, Hemophilia, Phenylketonuria, Cystic fibrosis, Sickle-cell anemia, Duchenne muscular dystrophy, Emphysema, Thalassemia
Genetically Modified Organisms (GMOs):
  • Organisms with genetic material that is artificially altered through genetic engineering.
  • Involves creating combinations of genes (plant, animal, bacterial, viral) that don't naturally occur or through traditional crossbreeding.
Transgenic Plants: Characteristics & Examples

  • Transgenic Plants: Genetically engineered to possess desirable traits.

Applications:

  • Insect pest resistance: e.g., Bt cotton, Transgenic tobacco.
  • Biofortification: Enhanced vitamins, proteins, oil, and iron.
  • Tolerance to abiotic stresses and herbicides.
  • Resistance to diseases.
  • Improvement in post-harvest characteristics: e.g., Flavr Savr tomatoes.

Examples:

  • Bt Cotton: Contains Bt toxin gene for insect resistance, reducing the need for insecticides.
  • Golden Rice: Rich in beta carotene (provitamin A) for better vitamin A intake.
  • Flavr Savr Tomato: Engineered to have longer shelf life by inhibiting polygalacturonase synthesis.

Insect-Resistant Transgenic Crops

 

 


 

Biofortified Transgenic Crops
  • Golden Rice & Golden Mustard: Enriched with vitamin A.
  • Soybean, Oil Palm, Rapeseed, Sunflower: Enhanced oil content and quality using Arabidopsis genes.
  • Ferritin-Rich Rice: Contains iron storage protein from soybean and Phaseolus for increased iron content.
  • Protein-Enriched Crops: Engineered to boost methionine, lysine, and tryptophan levels.
Transgenic Plants as Bioreactors
  • Production of biochemicals: Starch, sugar, lipids, proteins.
  • Biopharmaceuticals: Hormones, antibodies, vaccines, drugs, enzymes.
  • Manufacture of industrial products: Lubricants, biodegradable plastics, perfumes, adhesive compounds.
  • Renewable Energy Crops: Potential to replace fossil fuels.
  • Superglue: Produced by transgenic tobacco for use as biochemical glue in surgery.
  • Edible Vaccines: Delivered through modified potatoes, tomatoes, bananas, soybeans, alfalfa, and cereals for disease protection.
Transgenic Animals

  • Animals with a deliberate modification of their genome.
  • Produced using Recombinant DNA technology.
  • Foreign DNA is introduced and passed through the germ line, ensuring all cells contain modified genetic material.

Process:

  • Cloning of the desired gene.
  • Introduction of the cloned gene into fertilized eggs.
  • Successful implantation into a receptive female.
  • Progeny carry the cloned genes.

Applications of Transgenic Animals: Fields of Use: Medical Research, Toxicology, Molecular Biology, Pharmaceutical Industry 

Transgenic Mice and Cancer Research Purpose:

  • Used in cancer research to study relationships between oncogenes (cancer-causing genes) and cancer development.
Specific Research:
  • Transgenic mice with specific oncogenes develop particular cancers.
  • Developed models for breast cancer research at Harvard by Philip Leder.
  • Analyzed oncogenes myc and ras for their roles in breast cancer development.
Transgenic Farm Animals

Objectives:

  • Improve quality and quantity of milk, meat, and wool.
  • Increase egg production.
  • Develop disease-resistant animals.
  • Produce low-cost pharmaceuticals and biologicals.

Examples:

  • Transgenic Cattle: Enhanced food and therapeutic production.
  • Transgenic Sheep: Better quality wool and meat; also used as bioreactors.
  • Transgenic Pigs: Improved meat production, serve as bioreactors, and useful in xenotransplantation (human transplants).
  • Transgenic Chickens: Lower fat and cholesterol, high protein eggs, disease resistance, improved feed efficiency, and better meat quality.
Transgenic Fish
  • Commercially Important Fish: Species: Atlantic salmon, Catfish, Goldfish, Tilapia, Zebra-fish, Common carp, Rainbow trout.
  • Genetic Modifications: Introduced growth hormone, chicken crystalline protein, and E. coli hygromycin resistance gene.
  • Benefits: Increased cold tolerance and improved growth.
Bioethics
  • Definition: Bioethics: Study of moral vision, decisions, and policies regarding human behavior related to biological phenomena.
  • Role of Ethics: Regulates community behavior through a set of socially acceptable moral standards.
Key Areas of Bioethics (New Developments):
  • r-DNA Technology, Cloning, Transgenics, and Gene Therapy.
  • In Vitro Fertilization, Sperm Banks, Prenatal Genetic Selection, and Eugenics.
  • Euthanasia, issues surrounding death, and care for those in a comatose state.
  • Ethical considerations regarding the suffering of animals.
  • Concerns about the integrity of species due to transgenesis.
  • Gene transfer between humans and animals.
  • Risks of biotechnology to environment, health, and biodiversity.
Bioethical Concerns Related to GMOs

Environmental Effects:

  • Impact on non-target organisms and insect-resistant crops.
  • Gene flow and loss of biodiversity.
  • Concerns regarding the disruption of natural processes.

Biotechnology Ethics: General guidelines on what should and should not be done with recombinant DNA techniques.

Effects of Biotechnology

On the Environment

Herbicide Use and Resistance:

  • Hybrid strains of weeds may develop resistance through cross-pollination.
  • Example: Roundup-ready soybeans may confer resistance to neighboring plants.

Effects on Untargeted Species:

  • Bt corn can adversely affect non-target species like the Monarch butterfly.
  • Unintentional effects on neutral or beneficial species due to GM plants.

On Human Health

Allergic Reactions:

  • GMO crops may lead to unexpected allergic responses.
  • Example: Transgenic soybeans with Brazil nut genes caused allergies in those with nut allergies.

Long-Term Health Effects:

  • Unknown long-term impacts of GMOs due to their recent development.
  • Introduction of new proteins previously not ingested may have unforeseen effects.

Food Additives:

  • Potential to introduce nutrients, antibiotics, and vaccines.
  • Risk of creating antibiotic and vaccine-resistant strains of diseases.
Regulatory Measures
  • Stringent Regulations: Rapid advances in life sciences create bioethical challenges necessitating regulation.
Indian Government's Role: Genetic Engineering Approval Committee (GEAC):
  • Oversees the validity of research involving GMOs.
  • Addresses the safety of GMOs for public use.

Biopatent and Biopiracy

Patent: A patent is a special right granted by the government to an inventor.

Components:
  • Grant: Agreement with the inventor.
  • Specification: Subject matter of the invention.
  • Claims: Scope of the invention to be protected.
Biopatent
  • Definition: A biopatent is a biological patent for: Strains of microorganisms, Cell lines, Genetically modified organisms, DNA sequences, Biotechnological processes and products.
  • Rights: Allows patent holders to exclude others from: Making, Using, Selling, Importing the protected invention for a limited time.
  • Duration: Five years from the date of grant or seven years from the filing date, whichever is less.
  • Significance: Encourages innovation and promotes a scientific culture in society. Highlights the role of biology in shaping human society.
  • Examples: First biopatent: Genetically engineered bacterium Pseudomonas for oil spill cleanup. Patent on controlling plant gene expression (not granted in India) raised ethical concerns about food security and corporate monopolies.
Biopiracy
  • Definition: Biopiracy refers to the theft of natural products and obtaining patents without compensating the host country. Involves unauthorized misappropriation of biological resources and traditional knowledge.
Examples of Biopiracy

1. Patenting of Neem (Azadirachta indica):

  • USDA and American MNC W.R. Grace sought a patent for controlling pests using neem oil in the early 90s.
  • This case exemplifies biopiracy based on India's traditional knowledge.

2. Patenting of Basmati Rice:

  • Texmati is a trade name for Basmati rice patented by Texas-based Rice Tec Inc. in 1997.
  • Basmati is indigenous to the Indian subcontinent, making this a case of biopiracy.
  • USPTO rejected claims due to public opposition in March 2001.

3. Patenting of Haldi (Turmeric):

  • A patent was granted in March 1995 for healing properties of Haldi by American researchers of Indian origin.
  • This knowledge is traditional and not new; the CSIR requested a reexamination, leading to patent cancellation.