Semester 6
BP605T
Immunoblotting Techniques, Microbial Genetics & Biotransformation
This unit covers molecular biology techniques and microbial genetics essential for biotechnology: immunoblotting techniques (ELISA, Western blotting, Southern blotting), comparative genetic organization of prokaryotes and eukaryotes, mechanisms of horizontal gene transfer in bacteria (transformation, transduction, conjugation), the role of plasmids and transposons, microbial biotransformation for pharmaceutical production, and mutation types.
Syllabus & Topics
- 1ELISA (Enzyme-Linked Immunosorbent Assay): Most widely used immunoassay. Principle: antigen-antibody reaction + enzyme-labeled detection → color change proportional to analyte concentration. Types: (1) Direct ELISA: antigen coated on plate → enzyme-labeled primary antibody added → substrate → color. Simple but less sensitive. (2) Indirect ELISA: antigen coated → unlabeled primary Ab → enzyme-labeled secondary Ab → substrate → color. More sensitive (signal amplification). Used for: HIV screening (anti-HIV Ab), Hepatitis. (3) Sandwich ELISA: capture Ab coated → sample (antigen) added → detection Ab (enzyme-labeled) → substrate → color. Most specific. Used for: quantifying cytokines, hormones, proteins. (4) Competitive ELISA: sample antigen competes with labeled antigen for binding to antibody → higher sample concentration → less color.
- 2ELISA – Procedure & Applications: General procedure: (1) Coat 96-well microplate with antigen/antibody. (2) Block non-specific binding (BSA, casein). (3) Add sample. (4) Add enzyme-conjugated antibody. (5) Wash (remove unbound). (6) Add substrate (TMB for HRP, pNPP for ALP). (7) Stop reaction (H₂SO₄). (8) Read absorbance (spectrophotometer at 450 nm for TMB). Enzymes used: HRP (Horseradish Peroxidase), ALP (Alkaline Phosphatase). Applications: HIV testing (screening), Hepatitis B/C, COVID-19 antibody testing, pregnancy test (hCG), allergy testing (IgE), therapeutic drug monitoring, food safety (allergen detection), hormone levels (TSH, insulin).
- 3Western Blotting (Immunoblotting): Detects specific PROTEINS using antibodies. Steps: (1) SDS-PAGE: proteins separated by molecular weight in polyacrylamide gel (SDS denatures + gives uniform negative charge → separation by size only). (2) Transfer (blotting): proteins electroblotted from gel onto nitrocellulose/PVDF membrane (Western transfer). (3) Blocking: BSA/milk to prevent non-specific binding. (4) Primary antibody: specific for target protein → binds to it on membrane. (5) Secondary antibody: enzyme-conjugated (HRP/ALP) anti-species Ab → binds primary Ab. (6) Detection: chemiluminescence (ECL), colorimetric, or fluorescent substrate. Applications: confirmatory test for HIV (after ELISA screening), protein expression studies, detecting specific proteins in cell lysates.
- 4Southern Blotting: Developed by Edwin Southern (1975). Detects specific DNA sequences. Steps: (1) Restriction enzyme digestion: genomic DNA cut with RE → fragments. (2) Agarose gel electrophoresis: separate fragments by size. (3) Denaturation: NaOH → ssDNA. (4) Transfer: DNA transferred from gel to nylon/nitrocellulose membrane by capillary action (original) or vacuum/electroblotting. (5) Hybridization: radiolabeled or fluorescent DNA probe (complementary to target sequence) → binds to target DNA on membrane. (6) Detection: autoradiography or fluorescence imaging. Applications: gene detection, RFLP analysis (forensics, paternity testing, genetic disease diagnosis), detecting gene rearrangements (oncogenes). Related: Northern blotting = RNA detection (same principle, RNA separated by electrophoresis, detected with complementary probe).
- 5Genetic Organization – Prokaryotes vs Eukaryotes: Prokaryotes (E. coli): single circular chromosome (~4.6 Mb), no nuclear membrane (nucleoid), no histones, polycistronic mRNA (operon: lac, trp), no introns (genes are continuous), 70S ribosomes, plasmids (extrachromosomal DNA). Eukaryotes (humans): multiple linear chromosomes (23 pairs, ~3 billion bp), nuclear membrane, histones (chromatin), monocistronic mRNA, introns present (split genes: exons + introns → RNA splicing), 80S ribosomes, extensive post-transcriptional processing (capping, poly-A tail, splicing). Gene expression regulation: Prokaryotes — operons (lac operon: inducible). Eukaryotes — transcription factors, enhancers, silencers, epigenetics (methylation, histone modification).
- 6Microbial Genetics – Transformation: Horizontal gene transfer = transfer of genetic material between bacteria (not parent to offspring). Transformation: uptake of free/naked DNA from the environment. Discovered by Griffith (1928) — smooth vs rough Pneumococcus. Process: (1) Competent cell (naturally or artificially competent — CaCl₂ treatment, electroporation). (2) Double-stranded DNA binds to cell surface. (3) One strand degraded, complementary strand enters. (4) Integrates into chromosome by recombination. Natural competence: Bacillus, Haemophilus, Streptococcus, Neisseria. Artificial transformation: widely used in genetic engineering (plasmid DNA → competent E. coli).
- 7Transduction & Conjugation: Transduction: gene transfer mediated by bacteriophage. (1) Generalized transduction: during lytic cycle, any bacterial DNA fragment accidentally packaged into phage head → transferred to new host. P1 phage (E. coli). (2) Specialized transduction: lysogenic phage (λ) integrates near specific genes → during excision, takes adjacent bacterial genes → transfers only specific genes (gal, bio for λ). Conjugation: direct cell-to-cell contact through sex pilus (F pilus). Requires F factor (fertility plasmid). F⁺ (donor, has F plasmid) × F⁻ (recipient) → F pilus forms bridge → single strand of F plasmid transferred → F⁻ becomes F⁺. Hfr (High frequency recombination): F factor integrated into chromosome → during conjugation, chromosomal genes transferred (rarely complete transfer).
- 8Plasmids & Transposons: Plasmids: small, circular, self-replicating extrachromosomal DNA. Types: (1) F plasmid (fertility) — conjugation. (2) R plasmid (resistance) — antibiotic resistance genes (multiple drug resistance — clinical problem). (3) Col plasmid — colicin production (kills other bacteria). (4) Degradative plasmids — enzymes for unusual substrate degradation (toluene, camphor). (5) Ti plasmid (Agrobacterium) — used in plant genetic engineering. Copy number: stringent (1-2 copies — F plasmid) or relaxed (10-700 copies — pUC). Transposons (jumping genes): mobile genetic elements that can move (transpose) from one location to another in genome. Insertion sequences (IS): simplest — transposase gene flanked by inverted repeats. Composite transposons (Tn): carry additional genes (antibiotic resistance) flanked by IS elements. Significance: spread of antibiotic resistance, genome evolution, mutagenesis tools.
- 9Microbial Biotransformation: Use of microbial enzymes to chemically modify drug molecules. Advantages over chemical synthesis: stereoselective (produces specific enantiomer), regioselective, mild conditions, environmentally friendly. Types of reactions: (1) Oxidation (hydroxylation): Rhizopus converts Progesterone → 11α-hydroxyprogesterone (revolutionized steroid industry — replaced 37-step chemical synthesis with single microbial step). (2) Reduction: Corynebacterium reduces cortisone → hydrocortisone. (3) Hydrolysis: ester/amide hydrolysis. (4) Dehydrogenation: Arthrobacter → prednisolone from hydrocortisone. Applications: steroid transformations (most important — cortisone, prednisolone, dexamethasone), amino acid production (L-forms), antibiotic modifications, prodrug activation.
- 10Mutation – Types: Mutation: heritable change in DNA sequence. Spontaneous (natural replication errors) or Induced (mutagens: UV, chemicals, radiation). Types: (1) Point mutations: Single base substitution — Transition (purine↔purine or pyrimidine↔pyrimidine), Transversion (purine↔pyrimidine). Effects: Silent (same amino acid — degeneracy), Missense (different amino acid — sickle cell: GAG→GTG, Glu→Val), Nonsense (stop codon — truncated protein). (2) Frameshift: insertion or deletion of bases (not multiple of 3) → shifts reading frame → completely different protein downstream. (3) Large-scale: deletion, duplication, inversion, translocation of large DNA segments. Mutant types: Auxotrophic (require growth factor), Conditional (temperature-sensitive), Resistant (antibiotic/phage resistant). Ames test: uses Salmonella reverse mutation to detect mutagens/carcinogens.
Learning Objectives
ELISA Types: Compare direct, indirect, sandwich, and competitive ELISA with diagrams and applications.
Blotting Comparison: Differentiate Southern, Western, and Northern blotting by target molecule, probe, and procedure.
Gene Transfer: Compare transformation, transduction, and conjugation as gene transfer mechanisms in bacteria.
Biotransformation: Explain the contribution of microbial biotransformation to steroid drug production with examples.
Mutation Types: Classify mutations and explain the significance of the Ames test for carcinogen detection.
Exam Prep Questions
Q1. How do you remember which blotting technique detects what?
Memory trick: Southern (DNA) was named after Edwin Southern who invented it. Northern (RNA) was named as a play on Southern (RNA and DNA are both nucleic acids, so the “directional” naming). Western (Protein) continued the compass theme. So: SouDNA (Southern = DNA), Northern = RNA, Western = Protein. ELISA is different — it’s a plate-based immunoassay, not a blotting technique per se, but detects proteins/antibodies.
Q2. Why is ELISA used for HIV screening but Western blot for confirmation?
ELISA for HIV screening: high SENSITIVITY (catches almost all positives) but may give false positives (cross-reacting antibodies). It’s fast and inexpensive → ideal for screening large populations. Western blot for confirmation: high SPECIFICITY — separates HIV proteins by size, then detects antibodies against SPECIFIC HIV proteins (gp120, gp41, p24, etc.). A positive Western blot requires antibodies against multiple HIV proteins → virtually eliminates false positives. So: ELISA screens (catches all), Western blot confirms (eliminates false positives).
Q3. What was the significance of steroid biotransformation?
In 1952, Murray & Peterson discovered that the fungus Rhizopus could hydroxylate Progesterone at the 11α position in a SINGLE STEP. Previously, chemical synthesis required 37 steps. This biotransformation made cortisone and its derivatives (hydrocortisone, prednisolone, dexamethasone) commercially viable and affordable. It transformed the steroid pharmaceutical industry and demonstrated the enormous potential of microbial biotransformation for drug manufacturing.