Biotechnology and its Applications

Applications of Biotechnology

Biotechnology involves the use of living organisms or their components to create various products. Major applications include:
- Biopharmaceuticals: Production of medicinal drugs using living organisms.
- Therapeutics: Treatments for various diseases.
- Diagnostics: Methods for detecting diseases.
- Genetically Modified (GM) crops: Plants with altered genetic material used for agriculture.
- Processed food: Food products modified for better shelf life or nutrition.
- Bioremediation: Using organisms to clean up polluted environments.
- Waste treatment: Management of sewage and waste using biological processes.
- Energy production: Development of biofuels and other energy sources.

Research Areas in Biotechnology

There are $3$ critical research areas in the field of biotechnology:
- Providing the best catalyst: This involves selecting or creating an improved organism, typically a microbe or a specific enzyme.
- Creating optimal conditions: Using engineering principles to create the perfect environment for the biological catalyst to function effectively.
- Downstream processing technologies: Processes used to purify the resulting protein or organic compound from the mixture.

Increasing Food Production

To meet the demands of a growing population, there are $3$ primary options for increasing agricultural yield:
- Agro-chemical based agriculture: Relying on chemical fertilizers and pesticides. This method is expensive and causes significant environmental pollution.
- Organic agriculture: Farming without synthetic chemicals. This is also considered expensive.
- Genetically engineered crop-based agriculture: Utilizing crops that have been genetically modified for specific traits.

Genetically Modified Organisms (GMOs)

Definition: Genetically Modified Organisms are plants, bacteria, fungi, and animals whose genes have been altered through human manipulation.
Advantages of Genetic Modification in Plants:
- Abiotic Stress Tolerance: Makes crops more resilient to environmental factors such as cold, drought, salt, and heat.
- Pest Resistance: Reduces reliance on chemical pesticides as the plants themselves are resistant to pests.
- Reduced Post-harvest Losses: Improves the longevity and durability of crops after they are harvested.
- Mineral Usage Efficiency: Enhances the plant's ability to use soil minerals, which prevents the early exhaustion of soil fertility.
- Enhanced Nutritional Value: Improves the nutrient profile of food, such as Golden Rice, which is bio-fortified with Vitamin A.
- Tailor-made Plants: Creating specific plants to supply industries with alternative resources like starches, fuels, and pharmaceuticals.

Pest Resistant Plants: Bt Cotton

Pest resistant plants act as bio-pesticides, significantly reducing the need for chemical insecticides.
Examples include Bt cotton, Bt corn, rice, tomato, potato, and soybean.
Bacillus thuringiensis (Bt): Certain strains of this bacterium produce proteins that kill specific insects:
- Coleopterans: Beetles.
- Lepidopterans: Tobacco budworm, armyworm.
- Dipterans: Flies, mosquitoes.
Mechanism of Bt Toxin:
- B. thuringiensis forms an insecticidal protein (Bt toxin) crystal during a specific growth phase.
- The toxin exists as an inactive protoxin within the bacterium, so it does not kill the Bacillus.
- When an insect ingests the protoxin, the alkaline pH of the insect's gut solubilizes the crystals, activating the toxin.
- The active toxin binds to the surface of mid-gut epithelial cells, creating pores.
- This leads to cell swelling, lysis, and ultimately the death of the insect.
Specific Bt Genes:
- Bt toxin genes are isolated from B. thuringiensis and incorporated into crops like cotton.
- Most toxins are insect-group specific and are coded by cry genes.
- cryIAc and cryIIAb genes: Control cotton bollworms.
- cryIAb gene: Controls corn borer.

Nematode Resistance in Tobacco Plants via RNA Interference (RNAi)

The nematode Meloidogyne incognita infects tobacco plant roots, causing significant yield reduction.
Prevention is achieved through RNA interference (RNAi), a method of cellular defense present in all eukaryotic organisms.
RNAi Mechanism:
- It involves the silencing of a specific mRNA, preventing its translation.
- This silencing is caused by a complementary double-stranded RNA (dsRNA) molecule.
- The source of this dsRNA is often infection by RNA viruses or mobile genetic elements called transposons that replicate via an RNA intermediate.
Application in Tobacco:
- Nematode-specific DNA sequences are isolated.
- These are introduced into the host plant using Agrobacterium vectors.
- The DNA produces both sense and anti-sense RNA within the host cells.
- Because these RNAs are complementary, they form dsRNA.
- The dsRNA initiates RNAi, silencing the nematode's specific mRNA, meaning the parasite cannot survive in the transgenic host.

Biotechnology in Medicine

Recombinant DNA technology allows for the mass production of safe, effective therapeutic drugs.
Unlike products derived from non-human sources (which can cause immunological responses), recombinant therapeutics are generally well-tolerated.
Currently, approximately $30$ recombinant therapeutics are approved worldwide, with $12$ of these being marketed in India.

Genetically Engineered Insulin

Insulin is essential for managing adult-onset diabetes.
Traditionally, insulin was extracted from the pancreas of slaughtered cattle and pigs, which caused allergic reactions in some patients due to foreign protein sensitivities.
Insulin Structure:
- Consists of two short polypeptide chains: Chain A and Chain B.
- These chains are linked together by disulphide bridges.
Maturation Process:
- In mammals, insulin is synthesized as a pro-hormone (pro-insulin).
- It contains an extra stretch of protein called C peptide.
- Mature, functional insulin does not contain C peptide; it is removed during the maturation process.
Production in E. coli ( $1983$ ):
- The American company Eli Lilly prepared DNA sequences corresponding to human insulin chains A and B.
- These sequences were introduced into plasmids of E. coli.
- The A and B chains were produced separately, extracted, and then combined by creating disulfide bonds to form human insulin (Humulin).

Gene Therapy

Gene therapy is a technique used to correct gene defects identified in children or embryos.
It involves inserting functional genes into a person's cells or tissues to compensate for non-functional genes.
First Clinical Case ( $1990$ ):
- Administered to a $4$ -year old girl with adenosine deaminase (ADA) deficiency.
- ADA deficiency is caused by the deletion of the gene for the ADA enzyme, which is critical for immune system function.
Treatment Options for ADA:
- Bone marrow transplantation.
- Enzyme replacement therapy (ADA injection).
- Neither of the above are completely curative.
Gene Therapy Procedure for ADA:
- Lymphocytes are collected from the patient's blood and grown in a culture.
- A functional ADA cDNA is introduced into these lymphocytes using a retroviral vector.
- The modified lymphocytes are returned to the patient.
- Since lymphocytes are not immortal, the patient requires periodic infusions.
- Permanent Cure: Introducing the ADA gene from marrow cells into cells at the early embryonic stage could provide a permanent solution.

Molecular Diagnosis

Conventional methods like serum and urine analysis are often insufficient for early disease detection.
Polymerase Chain Reaction (PCR):
- Pathogens are usually only detected after symptoms appear, by which point their concentration is high.
- PCR allows for the detection of very low concentrations of bacteria or viruses by amplifying their nucleic acids.
- Uses of PCR:
  - Detecting HIV in suspected patients.
  - Detecting gene mutations in suspected cancer patients.
  - Identifying various genetic disorders.
- Autoradiography with Probes: A single-stranded DNA or RNA tagged with a radioactive molecule (probe) is hybridized to complementary DNA in a cell clone. Mutated genes will not appear on photographic film because the probe lacks complementarity with the mutation.
ELISA (Enzyme Linked Immuno-Sorbent Assay):
- Based on the principle of antigen-antibody interaction.
- Detects pathogens via specific antigens (proteins, glycoproteins) or by detecting antibodies produced by the host against the pathogen.

Transgenic Animals

These are animals that have had their genome altered by the introduction of a foreign gene.
Examples include transgenic rats, rabbits, pigs, sheep, cows, and fish.
Over $95\%$ of all existing transgenic animals are mice.
Benefits of Transgenic Animals:
- Studying Physiology and Development: Used to study how genes are regulated and how they affect normal body functions (e.g., studying insulin-like growth factor).
- Studying Human Diseases: Transgenic models serve as tools to understand how genes contribute to diseases and to test new treatments for cancer, cystic fibrosis, rheumatoid arthritis, and Alzheimer’s.
- Biological Products: Animals are engineered to produce complex human proteins.
  - Example: Human protein $\alpha$ -1-antitrypsin used to treat emphysema.
  - Other targets: Phenylketonuria (PKU) and cystic fibrosis.
  - Rosie ( $1997$ ): The first transgenic cow. She produced human protein-enriched milk ( $2.4\,g\,dm^{-3}$ ) containing human $\alpha$ -lactalbumin, making it more nutritionally balanced for human babies than natural cow milk.
- Vaccine Safety Testing: Transgenic mice are used to test the safety of the polio vaccine before use in humans, potentially replacing monkeys in these tests.
- Chemical Safety (Toxicity) Testing: Some transgenic animals are engineered to be more sensitive to toxic substances, allowing for immediate results upon exposure.

Ethical Issues and Biopiracy

Genetic modification can lead to unpredictable results, necessitating ethical oversight.
GEAC (Genetic Engineering Approval Committee): An Indian government organization that decides on the validity of GM research and the safety of introducing GM organisms for public services.
Biopiracy: The use of bio-resources by multinational companies or organizations without proper authorization from the countries or indigenous people concerned.
- Industrialized nations often possess superior technology but are poor in biodiversity and traditional knowledge.
- Developing nations are rich in biodiversity and traditional knowledge but may lack the means to exploit them.
Case Study: Basmati Rice:
- India has $27$ documented varieties of Basmati rice, known for its unique aroma.
- In $1997$ , an American company obtained patent rights on Basmati rice through the US Patent and Trademark Office.
- The company claimed to have created a "new" variety, but it was actually derived by crossing Indian Basmati with semi-dwarf varieties.
- This patent allowed the company to potentially restrict others from selling similar rice.
Other Biopiracy Examples: Attempts to patent traditional herbal medicines like Turmeric and Neem.
Indian Patents Bill: The Indian Parliament cleared a second amendment to this bill to address patent terms, emergency provisions, and research and development initiatives to prevent unauthorized exploitation.

Model Questions

1. There are many advantages of genetic modification in plants. Mention any four advantages.
2. Now a days Bt Brinjal has been much in the news. Being a GM Food is it advantageous or disadvantageous? List out any two points each.
3. Transgenic animals are said to be beneficial to humans. Justify this statement by giving two reasons.
4. Genetically modified tomato has some significance. Comment.
5. With an example, explain how biotechnology has been applied in each of the following:
- a) In curing Diabetes mellitus
- b) In rising pest resistant plants
- c) In producing nutritionally balanced milk.
6. Briefly explain the terms:
- a) cry gene
- b) C peptide
7. Explain RNA interference (RNAi) strategy.
8. Biotechnology has provided some techniques for early diagnosis of diseases. Mention any $2$ examples.
9. Expand the following abbreviations:
- a) GMO
- b) PCR
- c) ADA
- d) ELISA
- e) GEAC
1. Do you think it is ethical to manipulate organisms for human benefits? Justify your answer.
1. What do you understand by the term Biopiracy?