1.1 Microbiology: Organisms in Industry

Micro-organisms:

• Microorganisms are tiny, living organisms that are too small to be seen with the naked eye.

• Types: Include bacteria, viruses, fungi, protozoa, and algae.

• Found everywhere, in soil, water, air, and inside living organisms.

• Uses:

  • Decomposition: Break down organic matter.

  • Nutrient Cycling: Essential for recycling nutrients in ecosystems.

  • Food Production: Used in processes like fermentation for food and beverage production.

  • Medicine: Some microorganisms are used to produce antibiotics and vaccines.

Modes of Nutrition:

• Autotrophy:

  • Self-sufficient in nutrient production.

  •  Use inorganic sources to synthesize organic compounds.

  • Includes photoautotrophs and chemoautotrophs.

  • Examples: Photosynthesis in plants and some bacteria.

• Heterotrophy:

  •  Depend on external sources for organic nutrients.

  • Obtain carbon from organic compounds produced by other organisms.

  •  Includes saprotrophs (feed on dead matter) and parasites (derive nutrients from living hosts).

• Mixotrophy:

  • Combination of autotrophic and heterotrophic nutrition.

  • Can switch between modes depending on environmental conditions.

  • Examples: Certain algae and protozoa.

Modes of Respiration:

• Aerobic respiration:

  • Requires oxygen for the breakdown of organic compounds.

  • Yields more energy compared to other modes.

  • Common in many bacteria, fungi, and higher organisms.

• Anaerobic Respiration:

  • Takes place in the absence of oxygen.

  • Yields less energy compared to aerobic respiration.

  • Examples include fermentation in yeast and some bacteria.

• Facultative Anaerobes:

  • Can switch between aerobic and anaerobic respiration.

  • Adapt to the presence or absence of oxygen.

  • Common in versatile microorganisms.

• Obligate Anaerobes:

  •  Strictly anaerobic, cannot survive in the presence of oxygen.

  •  Found in environments lacking oxygen, such as deep sediments.

Gram Staining:

• It is a technique used to categorize bacteria into two groups based on cell wall characteristics.

• Steps:

  • Crystal Violet Staining: Applies purple dye to bacterial cells.

  • Iodine Treatment: Fixes the dye in the cells.

  • Alcohol Wash: Washes the dye from some cells, differentiating them.

  • Counterstaining with Safranin: Applies a red dye to the washed cells.

• Resulting Categories:

  • Gram-Positive: Retain the crystal violet dye, appearing purple.

  • Gram-Negative: Lose the dye, take on the safranin stain, appearing red.

• Cell Wall Differences:

  • Gram-Positive Bacteria: Thick peptidoglycan layer in the cell wall.

  • Gram-Negative Bacteria: Thin peptidoglycan layer, outer membrane.

Gram-positive V/S Gram-negative bacteria:

Gram-Positive Bacteria:

• Cell Wall: Thick peptidoglycan layer.

• Gram Stain Result: Retains crystal violet dye, appears purple.

• Outer Membrane: Absent.

• Toxins: Some produce exotoxins.

• Resistance to Antibiotics: Generally more susceptible.

• Examples: Staphylococcus, Streptococcus.

Gram-Negative Bacteria:

• Cell Wall: Thin peptidoglycan layer, outer membrane.

• Gram Stain Result:  Loses crystal violet dye, takes on safranin stain, appears red.

• Outer Membrane: Present, contains lipopolysaccharides.

• Toxins: May produce endotoxins.

• Resistance to Antibiotics: Often more resistant.

• Examples:  Escherichia coli, Salmonella.

Bactericides:

• Bactericides are substances or agents that kill bacteria or inhibit their growth.

• Modes of Action:

  • Disruption of cell membrane integrity.

  • Inhibition of essential metabolic processes.

  • Interference with cell wall synthesis.

• Common Types:

  • Antibiotics: Specific bactericides used in medical treatment.

  • Disinfectants: Bactericidal agents applied to surfaces or tissues.

  • Antiseptics: Bactericidal substances for use on living tissues.

Fermenters:

• Fermenters are controlled environments used for microbial fermentation processes.

• Microorganisms, such as bacteria or yeast, play a key role in fermenters.

• Fermentation Process: Microbes convert substrates (e.g., sugars) into products (e.g., alcohol or organic acids) in the absence of oxygen.

• Temperature, pH, oxygen levels, and nutrient concentrations are regulated for optimal microbial growth and product formation.

• Used in various industries, including food and beverage, pharmaceuticals, and biofuel production.

Microorganism Growth Rate:

Lag Phase:

• Initial period of adjustment.

• Cells adapt to the environment, synthesizing necessary enzymes.

Logarithmic (Log or Exponential) Phase:

• Rapid and exponential growth.

• Maximum reproduction and metabolic activity.

Stationary Phase:

• Growth rate slows, and reproduction equals cell death.

•  Nutrient depletion and accumulation of waste products.

Death Phase:

• Cell death exceeds reproduction.

• Decline in population due to resource exhaustion and waste toxicity.

Factors Affecting Growth Rate:

•  Nutrient availability.

•  Temperature and pH.

•   Oxygen levels.

•   Presence of inhibitors or toxins.

Batch vs Continuous Culture

Batch Culture:

• Nature: Closed system where a finite amount of medium is used.

• Operation: 

  • Inoculated with a fixed amount of microorganisms.

  •  No additions or removals during the process.

• Growth Phases: Exhibits distinct lag, log, stationary, and death phases.

• Applications: Small-scale experiments, laboratory studies, and certain industrial processes.

• Control:  Limited control over culture conditions during the process.

Continuous Culture:

• Nature: Open system with a continuous inflow of fresh medium.

• Operation:

  •  Ongoing addition of nutrients and removal of culture fluid. 

  • Allows for a steady-state condition.

• Growth Phases: Maintains cells in an exponential growth phase.

• Applications:  Large-scale industrial production, especially for constant product output.

• Control: Precise control over environmental factors like nutrient concentration and growth rate.

Pathway Engineering:

• Pathway engineering is a branch of synthetic biology that focuses on modifying or constructing biological pathways within organisms to produce desired compounds or perform specific functions.

• Modify existing pathways for enhanced production of a desired product.

• Construct new pathways to enable the synthesis of novel compounds.

• Optimize metabolic pathways for increased efficiency.

Biogas Production:

• Biogas is a clean and renewable fuel source.

• It is derived from organic matter such as agricultural waste, manure, sewage, and food waste.

•  It is produced through anaerobic digestion, a biological process involving bacteria that thrive in oxygen-free environments.

• Main Components of Biogas:

  • Methane (CH4): The primary combustible component, contributing to the energy value of biogas.

  • Carbon Dioxide (CO2:: Non-combustible by product, present in varying amounts.

  • Trace Gases: Small amounts of other gases, depending on feedstock and digestion conditions.

• Anaerobic Digestion Process:

  • Enzymatic Breakdown: Microbes break down organic matter into simpler compounds.

  • Methanogenesis: Methane-producing bacteria convert intermediates into methane and carbon dioxide.

  • Temperature and pH: Optimal conditions vary, typically mesophilic (room temperature) or thermophilic (higher temperatures) digestion.

• Feedstock Sources:

  • Agricultural Residues

  • Municipal Waste

  • Manure.

• Benefits of Biogas Production:

  • Renewable Energy

  • Waste Management

  • Reduced Greenhouse Gas Emissions

• Working: A biogas plant works by harnessing the natural process of anaerobic digestion. 

1. Feedstock Collection: Organic waste materials like agricultural residues, animal manure, or food waste are collected and fed into the plant.

2. Digester: The waste is placed in an airtight container called a digester, where it undergoes decomposition in the absence of oxygen.

3. Anaerobic Digestion: Bacteria and other microorganisms break down the organic matter in the digester, producing biogas as a byproduct. This biogas is primarily composed of methane and carbon dioxide.

4. Biogas Storage: The produced biogas is collected and stored in a tank for further use.

5. Biogas Utilization: Biogas can be used directly for cooking and heating or converted into electricity through a gas engine. The remaining digested material, called digestate, can be used as a fertilizer.

                                                       Biogas plant

1.2 Biotechnology in Agriculture

Transgenics:

• It involves the introduction of foreign genes into an organism's genome, creating genetically modified organisms (GMOs).

• The purpose is to confer specific traits or characteristics that are not naturally present in the organism.

• Genetic Modification Process:

  • Isolation of Gene: Identification and isolation of the gene with the desired trait.

  • Vector Use: Insertion of the gene into a vector (often a plasmid or viral DNA).

  • Transformation: Introduction of the vector into the target organism's cells.

  • Integration: Incorporation of the foreign gene into the host organism's genome.

Genetic Identification:

• The process of pinpointing the location and function of genes within a DNA sequence.

• It is very essential for understanding genetic traits, disease susceptibility, and biological functions.

• Techniques for Gene Identification:

  • PCR (Polymerase Chain Reaction): Amplifies specific DNA segments for further analysis.

  • DNA Sequencing: Determines the order of nucleotides in a DNA molecule, aiding in gene identification.

  • Microarray Analysis: Examines gene expression patterns to identify active genes.

  • CRISPR-Cas9 Technology: Enables targeted gene editing and functional analysis.

• Bioinformatics Tools:

  • Gene Prediction Algorithms: Computational methods to identify potential gene locations based on DNA sequences.

  • Comparative Genomics: Comparing genomes of different species to identify conserved gene regions.

  • Functional Genomics: Analyzing gene functions through large-scale data analysis.

• Controlling Expression of a Target Gene:

Gene Delivery Systems:

• Methods and technologies for introducing genetic material into target cells for therapeutic or research purposes.

• It facilitates the transfer of genes for gene therapy, genetic engineering, or research studies.

• Viral Gene Delivery (Vector Delivery):

  • Adenoviruses and Lentiviruses: Modified viruses used as vectors to deliver genes into host cells.

  • Advantages: High efficiency in gene transfer, especially in dividing cells.

  • Challenges: Potential immunogenicity and safety concerns.

• Non-Viral Gene Delivery (Chemical Delivery):

  • Liposomes and Lipid Nanoparticles: Lipid-based carriers that encapsulate and transport genetic material.

  • Polymer-Based Delivery Systems: Polymers used to form complexes with DNA for cellular uptake.

  • Advantages: Reduced immunogenicity and safety concerns.

  • Challenges: Generally lower efficiency compared to viral methods.

• Physical Methods:

  • Electroporation: Brief electrical pulses create temporary pores in cell membranes for gene entry.

  • Gene Gun (Biolistics): Accelerates genetic material-coated particles into target cells using high pressure.

  • Microinjection: A fine needle is used to directly inject the recombinant DNA into a single host cell.

  • Advantages: Non-viral, applicable to various cell types.

  • Challenges: Variable efficiency and potential cell damage.

• Applications:

  • Gene therapy

  • Research and functional genomics

  • Vaccine development

Gene Expression:

• Following gene delivery, scientists must identify which cells have successfully taken up the recombinant DNA

• These transgenic cells can then be cultured and developed to form transgenic crops for agricultural use

• Marker genes are typically included as part of the recombinant construct to indicate successful uptake

GM Crops:

• Plants whose genetic material has been altered through genetic engineering techniques.

• The purpose is to introduce specific traits for improved yield, resistance, or nutritional content.

• Common Traits in GM Crops:

  • Pest Resistance: Incorporation of genes to deter or resist pests.

  • Herbicide Tolerance: Genes enabling resistance to specific herbicides.

  • Disease Resistance: Enhancement of resistance to certain diseases.

  • Nutritional Enhancement: Modification for improved nutritional content.

• Popular GM Crops:

  • Bt Cotton: Expresses Bacillus thuringiensis toxin for insect resistance.

  • Roundup Ready Soybeans: Tolerant to glyphosate herbicides.

  • Golden Rice: Engineered to produce beta-carotene, a precursor of vitamin A.

• Benefits:

  • Increased Crop Yields: Resistance to pests and diseases can enhance overall productivity.

  • Reduced Need for Pesticides: Pest-resistant crops may reduce reliance on chemical pesticides.

  • Improved Nutritional Content: Biofortified crops address nutritional deficiencies.

• Challenges and Concerns:

  • Environmental Impact: Potential effects on non-target organisms and ecosystems.

  • Resistance Management: Development of resistance in pests to transgenic traits.

  • Gene Flow: Transfer of transgenes to wild relatives or nearby crops.

  • Consumer Acceptance: Public concerns about the safety and ethics of GMOs.

• Regulation:

  • Varied Standards: Different countries have different regulations governing the cultivation and consumption of GM crops.

  • Risk Assessment: Rigorous evaluations to assess potential environmental and health risks.

1.3 Biotechnology in Environment

Bioremediation:

• Application of biological processes to eliminate or neutralize pollutants in the environment.

• Biotechnology in Bioremediation:

  • Genetically Engineered Microorganisms (GEMs): Modified microbes designed for enhanced pollutant degradation.

  • Enzyme Technology: Using enzymes for targeted degradation of specific pollutants. 

• Types of Bioremediation:

  • Phytoremediation: Use of plants to absorb, accumulate, or transform contaminants in soil or water.

  • Microbial Remediation: Employing microorganisms like bacteria, fungi, or algae to break down pollutants.

  • Bioaugmentation: Introducing specific microorganisms to enhance natural degradation processes.

• Advantages of Bioremediation:

  • Environmentally Friendly

  • Cost-Effective

  • Sustainable

• Applications:

  • Land Decontamination: Treating soil polluted by industrial activities or spills.

  • Water Purification: Cleaning contaminated water bodies and groundwater.

  • Oil Spill Cleanup: Accelerating the natural degradation of oil spills.

Pollutant Metabolism:

• The biochemical modification of pollutants, often making them less harmful or easier to eliminate from the body.

  • A pollutant is a substance introduced into the environment that has an adverse effect upon a natural resource.

• Key Players:

  • Microorganisms: Bacteria, fungi, and algae often play a major role in breaking down pollutants in the environment.

  • Enzymes: These proteins within living organisms catalyze reactions that transform pollutants.

• Mechanisms:

  • Oxidation: Addition of oxygen to the pollutant molecule.

  • Reduction: Removal of oxygen or addition of hydrogen.

  • Hydrolysis: Breakdown of a compound by reaction with water.

  • Conjugation: Combining the pollutant with another substance to make it more water-soluble.

• Outcomes:

  • Detoxification: Pollutant is rendered less harmful.

  • Bioactivation: Pollutant is transformed into a more toxic substance (less common).

  • Biodegradation: Pollutant is completely broken down into harmless components.

• Factors Influencing Metabolism:

  • Pollutant type: Chemical structure and properties determine how it can be metabolized.

  • Organism: Different species have varying metabolic capabilities.

  • Environmental conditions: Temperature, pH, and nutrient availability can affect metabolism.

• Examples:

  • Oil spills: Microorganisms can break down hydrocarbons in oil.

  • Pesticide degradation: Some pesticides are degraded by soil microbes.

  • Heavy metal transformation: Microbes can change the oxidation state of heavy metals, affecting their toxicity and mobility.

• Applications:

  • Bioremediation: Using living organisms to clean up polluted environments.

  • Environmental monitoring: Assessing the metabolic potential of an environment to predict pollutant fate.

• Challenges:

  • Complex mixtures: Many pollutants exist as mixtures, making metabolism difficult to predict.

  • Emerging pollutants: New pollutants are constantly being released, requiring research into their metabolism.

  • Long-term effects: Some pollutants or their metabolites may have persistent effects on ecosystems.

Biofilms:

• Biofilms are complex communities of microorganisms (bacteria, fungi, algae, etc.) attached to a surface and embedded in a self-produced matrix of extracellular polymeric substances (EPS).

• Ubiquitous: Biofilms are found in almost every environment, from natural settings (rivers, oceans, soil) to man-made structures (pipes, medical implants).

• Formation: Biofilms develop in stages, starting with the initial attachment of planktonic (free-floating) cells to a surface, followed by growth, maturation, and dispersion.

• Properties:

  • Resistance: Biofilms are highly resistant to antibiotics, disinfectants, and the host's immune system.

  • Communication: Microorganisms within biofilms communicate through quorum sensing, a chemical signaling process.

  • Diversity: Biofilms can contain a variety of species, each contributing different functions.

• Impacts:

  • Positive: Biofilms can be beneficial in wastewater treatment and bioremediation.

  • Negative: Biofilms can cause chronic infections (e.g., cystic fibrosis), dental plaque, and corrosion of industrial equipment.

• Research: Biofilms are an active area of research due to their significance in medicine, industry, and the environment.

Biofilm Development Process:

1. Initial Attachment: Planktonic cells reversibly attach to a surface.

2. Irreversible Attachment: Cells adhere more strongly through adhesins and start producing EPS.

3. Maturation I: Microcolonies form, and the biofilm structure begins to develop.

4. Maturation II: The biofilm matures with the formation of channels for nutrient and waste transport.

5. Dispersion: Some cells detach from the biofilm and become planktonic again, colonizing new surfaces.

Quorum Sensing

• As the biofilm develops, microorganisms will alter their gene expression in response to changes in population density

  • This pattern of behaviour is called quorum sensing and is mediated by the release of chemicals from colony cells.

  • Quorum sensing allows the biofilm to grow and develop in a manner that is not uniform and promotes structural stability.

Use of Biofilms:

Use of Biofilms in Trickle Filter Beds for Sewage Treatment:

• Trickle Filters: Beds of rocks or other media over which wastewater is sprayed.

• Biofilm Formation: A slimy layer of diverse microorganisms forms on the media surface.

• Wastewater Treatment:

  • Organic Matter Degradation: Biofilm microbes break down organic pollutants in the wastewater.

  • Nitrification: Some bacteria convert ammonia to nitrite and nitrate (less toxic forms).

  • Denitrification: Other bacteria convert nitrate to nitrogen gas (removed from the system).

  • Suspended Solids Removal: Biofilm traps and removes some solids from the wastewater.

• Advantages of Biofilms:

  • High Efficiency: Biofilms have a large surface area for microbial activity, increasing treatment capacity.

  • Resilience: Biofilms are resistant to shock loads and toxic substances, providing stable treatment.

  • Cost-Effective: Trickle filters are relatively simple and inexpensive to operate.

• Maintenance:

  • Sloughing: Periodic removal of excess biofilm to prevent clogging and maintain efficiency.

  • Backwashing: Flushing the filter bed with water to remove debris and excess biomass.

Bacteriophages:

• Viruses that specifically infect and replicate within bacteria. Often called "phages."

• Abundance: Most abundant biological entities on Earth, found wherever bacteria exist.

• Structure:

  • Head: Contains genetic material (DNA or RNA).

  • Tail: Attaches to bacteria and injects genetic material.

  • Other components: May have additional structures like tail fibers or baseplate.

• Life Cycle:

  • Lytic cycle: Phage infects, replicates, and kills the host bacterium.

  • Lysogenic cycle: Phage DNA integrates into the bacterial genome (prophage), replicating with the host until it enters the lytic cycle.

• Diversity:

  • Morphology: Various shapes and sizes (e.g., icosahedral, filamentous, head-tail).

  • Genetic diversity: Wide range of genetic material and genomic organization.

  • Host specificity: Phages can be highly specific to certain bacterial strains.

• Applications:

  • Phage Therapy: Using phages to treat bacterial infections (alternative to antibiotics).

  • Food Safety: Controlling foodborne pathogens (e.g., Listeria, Salmonella).

  • Biotechnology: Tools for genetic engineering, diagnostics, and research.

• Challenges:

  • Narrow host range: Limited effectiveness against diverse bacterial populations.

  • Bacterial resistance: Bacteria can develop resistance to phages.

  • Regulatory hurdles: Approval for phage therapy is complex and varies between countries.

1.4 Biotechnology in Medicine

Disease Markers:

• Disease markers are measurable substances or characteristics that indicate the presence, severity, or risk of a disease. They can be used for diagnosis, prognosis, and monitoring treatment response.

• These foreign markers which are capable of eliciting an immune response are called antigens.

• The part of the antigen to which specific antibodies will bind is called an epitope (or antigenic determinant).

There are three main types of disease markers:

1. Surface Markers:

• Molecules found on the surface of cells or tissues.

• Examples:

  • Proteins (receptors, antigens)

  • Carbohydrates (sugars)

  • Lipids (fats)

• Detection:

  • Immunohistochemistry (IHC)

  • Flow cytometry

• Applications:

  • Cancer diagnosis (tumor markers)

  • Immune system disorders (CD markers)

  • Infectious diseases (viral or bacterial antigens)

2. Genetic Markers:

• Alterations in DNA sequence or gene expression.

• Examples:

  • Single nucleotide polymorphisms (SNPs)

  • Mutations in specific genes

  • Changes in gene copy number

• Detection:

  • Polymerase chain reaction (PCR)

  • DNA sequencing

  • Microarray analysis

• Applications:

  • Inherited diseases (cystic fibrosis, sickle cell anemia)

  • Cancer predisposition (BRCA1/2 mutations)

  • Pharmacogenomics (personalized medicine)

3. Metabolic Markers:

• Substances produced or used by the body during metabolism.

Blood glucose

• Examples:

  • Glucose (diabetes)

  • Cholesterol and triglycerides (heart disease)

  • Liver enzymes (liver damage)

  • Hormones (thyroid disorders)

• Detection:

  • Blood tests

  • Urine tests

• Applications:

  • Diabetes diagnosis and monitoring

  • Cardiovascular disease risk assessment

  • Liver and kidney function tests

DNA Microarrays:

• A collection of microscopic DNA spots attached to a solid surface (e.g., glass slide, silicon chip).

• To measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.

• Technology:

  • Spots: Each spot contains thousands of identical copies of a specific DNA sequence (probe), which is complementary to a particular gene or DNA sequence of interest.

  • Hybridization: Fluorescently labelled DNA or RNA from a sample (target) is hybridized (bound) to the probes on the microarray.

  • Detection: The intensity of the fluorescent signal at each spot indicates the amount of target that hybridized to the probe, reflecting the expression level or presence of the corresponding gene or sequence.

• Gene Expression Profiling: Measuring the expression of thousands of genes in different conditions (e.g., healthy vs. diseased tissue, different stages of development) to identify differentially expressed genes and potential biomarkers.

• Genotyping: Detecting variations in DNA sequence (e.g., single nucleotide polymorphisms - SNPs) associated with diseases or traits.

• Comparative Genomic Hybridization (CGH): Identifying chromosomal abnormalities (deletions, duplications) in cancer cells or genetic disorders.

• Drug Development: Screening potential drug targets and evaluating drug efficacy and toxicity.

• Toxicology: Assessing the effects of environmental toxins on gene expression.

• Advantages:

  • High Throughput: Allows simultaneous analysis of thousands of genes or sequences.

  • Sensitivity: Can detect low levels of gene expression or DNA.

  • Versatility: Can be used for a wide range of applications.

cDNA Microarrays:

• cDNA (complementary DNA): DNA synthesized from mRNA, representing the expressed genes in a sample.

• cDNA Microarrays: Contain thousands of cDNA spots representing different genes.

• Hybridization: Fluorescently labeled cDNA from a patient's sample is hybridized to the microarray.

• Analysis: Detects changes in gene expression patterns associated with diseases.

PCR Analysis:

• A laboratory technique used to amplify specific DNA sequences to detectable levels.

• Purpose:

  • Detection: Identify the presence or absence of specific DNA sequences (e.g., pathogens, genetic mutations).

  • Quantification: Measure the amount of DNA or RNA present (e.g., viral load, gene expression levels).

  • Amplification: Generate large quantities of DNA for further analysis (e.g., sequencing, cloning).

• Components:

  • DNA template: The DNA sequence to be amplified.

  • Primers: Short, single-stranded DNA sequences that bind to the target DNA and initiate replication.

  • DNA polymerase: Enzyme that synthesizes new DNA strands using the template and primers.

  • Nucleotides: Building blocks for new DNA strands.

• Steps:

  • Denaturation: Heating the DNA to separate the two strands.

  • Annealing: Cooling the DNA to allow primers to bind to complementary sequences on the template.

  • Extension: DNA polymerase synthesizes new DNA strands, starting from the primers.

  • Cycling: Repeating the denaturation, annealing, and extension steps multiple times to exponentially amplify the target DNA.

  • Environmental monitoring: Identifying microorganisms in water and soil samples.

• Variations:

  • Real-time PCR (qPCR): Measures the accumulation of DNA in real time, allowing for quantification.

  • Reverse transcription PCR (RT-PCR): Amplifies RNA by first converting it to cDNA using reverse transcriptase.

  • Digital PCR (dPCR): Provides absolute quantification of DNA molecules by partitioning the sample into thousands of individual reactions.

ELISA:

• An enzyme-linked immunosorbent assay (ELISA) is a test that uses enzymes and color changes to identify a substance.

• Principle: Utilizes the specific interaction between antigens and antibodies.

• Steps:

  • Coating: Immobilizing the antigen or antibody of interest on a solid surface (e.g., microplate well).

  • Blocking: Preventing non-specific binding of antibodies to the surface.

  • Sample Addition: Adding the sample containing the target antigen or antibody.

  • Incubation: Allowing the antigen-antibody complex to form.

  • Washing: Removing unbound substances.

  • Detection: Adding an enzyme-linked antibody that binds to the target.

  • Substrate Addition: Adding a substrate that reacts with the enzyme, producing a color change.

  • Measurement: Quantifying the color change using a spectrophotometer, which is proportional to the amount of target substance present.

• Advantages:

  • High Sensitivity: Can detect minute amounts of target substances.

  • Specificity: Can distinguish between closely related antigens or antibodies.

  • Versatility: Can be adapted to detect a wide range of substances.

  • Easy to Perform: Relatively simple and inexpensive technique.

Tracking Experiments:

• If a protein is labelled to enable detection, its subsequent movements can be tracked to gain information about its function.

• Proteins can be tracked by tagging with a fluorescent molecule to enable detection with appropriate imaging devices.

• Example: it can be used to track tumor cells.

Biopharming:

• Involves using transgenic plants or animals to produce (or ‘farm’) pharmaceutical products for therapeutic use

  • This involves the insertion of target genes into hosts (crops or animals) that would not normally express those genes

  • The desired compound can potentially be expressed in a form that is routinely harvested (e.g. milk, eggs, fruits, etc.)

Gene Therapy:

• The introduction of genetic material into a patient's cells to replace or repair faulty genes or to introduce new therapeutic genes.

• Aims to treat or cure diseases caused by genetic defects.

• Types:

  • Somatic Gene Therapy:

    • Targets non-reproductive (somatic) cells.

    • Changes are not inherited by offspring.

  • Germline Gene Therapy:

    • Targets reproductive cells (sperm or eggs).

    • Changes are passed on to future generations (currently highly controversial and not approved for use in humans).

• Methods of Delivery:

  • Viral Vectors:

    • Modified viruses used as vehicles to deliver therapeutic genes into cells.

    • Common vectors include adenoviruses, retroviruses, lentiviruses, and adeno-associated viruses (AAVs).

• Non-Viral Vectors:

  • Liposomes (lipid-based nanoparticles)

  • Naked DNA

  • Other nanoparticles

• Applications:

  • Inherited Genetic Disorders:

    • Cystic fibrosis

    • Sickle cell anemia

    • Haemophilia

    • Muscular dystrophy

  • Cancer:

    • Immunotherapy (e.g., CAR-T cell therapy)

    • Oncolytic virus therapy

  • Infectious Diseases:

    • HIV/AIDS

• Challenges:

  • Safety:

    • Immune response to viral vectors

    • Potential for insertional mutagenesis (disrupting other genes)

    • Off-target effects

  • Efficacy:

    • Efficient delivery of therapeutic genes to target cells

    • Long-term expression of the introduced genes

  • Ethical Considerations:

    • Germline gene therapy raises concerns about genetic manipulation of future generations.

    • Equity and access to expensive treatments.

1.5 Bioinformatics

• Bioinformatics is an interdisciplinary field that combines biology, computer science, statistics, and information technology to analyze and interpret biological data. It involves the development and application of computational tools and algorithms to understand and utilize complex biological information.

Databases Used in Bioinformatics:

• GenBank: A comprehensive database of DNA sequences from various organisms.

• Protein Data Bank (PDB): A repository of protein structures determined by X-ray crystallography, NMR spectroscopy, and other methods.

• Gene Expression Omnibus (GEO): A database of gene expression profiles from various organisms and experimental conditions.

Ensemble: A genome browser for visualizing genomic data.

Chromosome 21: 

• Smallest human chromosome: Contains about 1.5% of the total DNA in cells.

• Location: One of the 23 pairs of chromosomes found in human cells.

• Gene Content: Estimated to contain 200-300 genes.

• Significance:

  • Trisomy 21: Three copies of chromosome 21 cause Down syndrome, the most common chromosomal condition.

  • Other disorders: Changes in the number or structure of chromosome 21 can lead to various health issues, including intellectual disability and developmental delays.

Gene discovery:

• Comparative genomics is a field of bioinformatics in which the genome sequence of different species is compared.

• Importance:

  • Understanding biological functions and processes.

  • Identifying disease-causing genes.

  • Developing new diagnostic tools and therapies.

• Methods:

  • Traditional:

    • Linkage analysis (studying inheritance patterns).

    • Positional cloning (mapping genes based on their location on chromosomes).

  • Modern:

    • DNA sequencing (determining the order of nucleotides in DNA).

    • Bioinformatics (analyzing and interpreting large datasets of genomic information).

    • Functional genomics (studying the function of genes and their interactions).

• Challenges:

  • The human genome is vast and complex.

  • Many genes have unknown functions.

  • Some genes are rare or difficult to detect.

Sequence alignment:

• The process of arranging two or more sequences (DNA, RNA, or protein) to identify regions of similarity.

• Aims to determine the degree of relatedness and identify functional, structural, or evolutionary relationships.

• Types:

  • Pairwise Alignment: Comparing two sequences.

  • Multiple Sequence Alignment (MSA): Comparing three or more sequences.

  • Global Alignment: Aligning entire sequences from end to end.

  • Local Alignment: Aligning specific regions with the highest similarity.

• Methods:

  • Needleman-Wunsch algorithm: Global alignment algorithm based on dynamic programming.

  • Smith-Waterman algorithm: Local alignment algorithm based on dynamic programming.

  • Progressive alignment (e.g., Clustal): Aligning multiple sequences by progressively adding them to an existing alignment.

• Scoring:

  • Substitution matrices: Assigns scores to matches, mismatches, and gaps based on evolutionary relationships.

  • Gap penalties: Penalties for introducing gaps in the alignment.

  • Scoring functions: Combine substitution matrix scores and gap penalties to calculate the overall alignment score.

• Tools:

  • BLAST: Basic Local Alignment Search Tool, used for searching databases for similar sequences.

  • Clustal Omega: Popular tool for multiple sequence alignment.

  • MAFFT: Another widely used tool for multiple sequence alignment.

  • MUSCLE: Fast and accurate tool for multiple sequence alignment.

Phylogenetics:

• The study of evolutionary relationships among organisms.

• To reconstruct the evolutionary history of life and understand how different species are related to each other.

• Data Sources:

  • Morphological: Physical traits and characteristics of organisms.

  • Molecular: DNA, RNA, and protein sequences.

• Methods:

  • Phylogenetic Trees: Graphical representations of evolutionary relationships, often depicted as branching diagrams.

  • Cladistics: A method that groups organisms based on shared derived characteristics.

  • Maximum Likelihood: A statistical method that evaluates the probability of different evolutionary scenarios.

Construction of Phylogeny tree:

1. Data: Gather morphological (traits) or molecular (DNA/RNA) data.

2. Alignment: (If molecular) Align sequences to match corresponding positions.

3. Distance: Calculate evolutionary distances between organisms.

4. Tree Building: Use an algorithm (e.g., Neighbor-Joining, Maximum Parsimony) to construct the tree based on distances.

5. Evaluation: Assess the tree's reliability using statistical methods.

6. Visualization: Draw a branching diagram, labeling tips and indicating evolutionary distance with branch lengths.

Gene Knockout:

• A genetic technique in which a specific gene is inactivated or removed from an organism's genome.

• Also known as gene targeting or gene deletion.

  • To study the function of a gene by observing the phenotypic effects of its absence.

  • To model human diseases caused by gene mutations.

  • To develop potential therapies for genetic disorders.

• Methods:

  • Homologous Recombination:

    • Replacing the target gene with a modified version that is non-functional.

    • Often used in mice to create knockout mouse models.

  • CRISPR-Cas9:

    • A more recent and efficient gene editing tool.

    • Can be used to create precise deletions or mutations in a gene.