CHSE Odisha Elective Biotechnology cheat sheet

Biotechnology definition (CBD-style): any technological application using biological systems, living organisms or derivatives to make or modify products or processes for specific use.

Pasteur (1857) disproved spontaneous generation; Buchner (1897) showed cell-free yeast extract ferments sugar, founding modern biochemistry.

Escherichia coli K-12 recombinant DNA era: Cohen and Boyer (1973) produced first recombinant plasmid; Sanger dideoxy sequencing 1977.

Khorana synthesized a gene chemically (1970); Karry Mullis invented PCR (1983); first recombinant human insulin approved 1982.

Colour code of biotechnology: red = medical, green = agricultural, blue = marine, white/grey = industrial, yellow = food, brown = arid/desert, dark = bioterrorism/biowarfare.

Indian milestones: Department of Biotechnology (DBT) set up 1986; National Biotechnology Board 1982; first transgenic crop approved (Bt cotton, 2002).

Odisha: Institute of Life Sciences (Bhubaneswar) is a DBT autonomous institute; Odisha University of Agriculture and Technology and Ravenshaw University offer biotechnology programmes.

Scale ladder in bioprocess: shake flask < bench fermenter < pilot < production; each 10-fold step needs re-validation of kLa and mixing time.

Prokaryote: 70S ribosome (50S+30S), no membrane-bound organelles, single circular chromosome, peptidoglycan wall; eukaryote: 80S ribosome (60S+40S).

Mitochondrial electron transport complexes I-IV plus ATP synthase (complex V); chemiosmotic theory (Mitchell) couples proton gradient to ATP synthesis.

Cell cycle checkpoints: G1/S (restriction point, Rb-E2F), G2/M (cyclin B-CDK1), spindle assembly checkpoint (Mad2).

Apoptosis is caspase-dependent and ATP-requiring; necrosis is passive swelling; autophagy is lysosome-mediated recycling.

Fluid mosaic model (Singer and Nicolson, 1972): phospholipid bilayer with integral and peripheral proteins; cholesterol modulates fluidity.

Osmosis: water moves to higher solute potential; plasmolysis in plant cells occurs in hypertonic solution, turgor is lost; wall pressure opposes osmotic pressure.

Plant cell unique structures: cell wall (cellulose), large central vacuole, chloroplast, plasmodesmata; animal cells have centrioles and lysosomes.

Bacterial growth phases: lag, log (exponential), stationary, death; generation time g = t/n and specific growth rate µ = 0.693/g.

Batch growth yield: Yx/s = biomass produced / substrate consumed; Monod equation µ = µmax·S/(Ks+S).

Sterilisation decision rule: autoclave 121 °C, 15 psi, 15-20 min for media; 0.22 µm filtration for heat-labile liquids; dry heat 160 °C/2 h for glassware.

Gram staining sequence: crystal violet, iodine (mordant), alcohol decolouriser, safranin counterstain; Gram-negative pink, Gram-positive purple.

Pure culture methods: streak plate, pour plate, spread plate; preservation by lyophilisation, glycerol stock at -80 °C, or subculture on slants.

Enumeration: viable plate count (CFU/mL = colonies / (dilution × volume plated)), turbidity (OD600), most probable number for coliforms.

Growth control: bacteriostatic inhibits growth, bactericidal kills, bacteriolytic lyses; minimum inhibitory concentration determined by broth dilution.

DNA double helix (Watson-Crick, 1953): antiparallel strands, A=T two H-bonds, G≡C three H-bonds; B-DNA 10 bp/turn, 3.4 nm pitch, 0.34 nm rise.

Chargaff rules: %A=%T, %G=%C; (A+G)=(T+C); GC content correlates with melting temperature.

Replication is semiconservative (Meselson-Stahl), bidirectional from ori, needs helicase, primase, DNA pol III (5'→3' synthesis, 3'→5' proofreading), ligase joins Okazaki fragments.

Transcription: RNA polymerase reads template 3'→5' and synthesises RNA 5'→3'; promoters (TATA box in eukaryotes, -10/-35 in bacteria) set direction.

Eukaryotic mRNA processing: 5' cap (7-methylguanosine), 3' poly-A tail, splicing out introns by spliceosome.

Genetic code: triplet, degenerate, non-overlapping, universal; 64 codons, 61 sense, 3 stop (UAA, UAG, UGA); AUG is start and codes methionine.

Translation: initiation (Shine-Dalgarno in bacteria, Kozak in eukaryotes), elongation (A, P, E sites), termination by release factors; central dogma DNA→RNA→protein.

Restriction enzymes: Type II cut palindromic sequences producing sticky or blunt ends; EcoRI GAATTC, BamHI GGATCC, HindIII AAGCTT, SmaI CCCGGG (blunt).

Cloning vector essentials: origin of replication, selectable marker, multiple cloning site, promoter; plasmid, phage, cosmid, BAC, YAC for larger inserts.

Ligation: T4 DNA ligase joins compatible ends; alkaline phosphatase prevents self-ligation; linkers/adaptors add restriction sites.

Transformation methods: CaCl2 heat shock, electroporation, microinjection, biolistic gene gun, Agrobacterium tumefaciens Ti plasmid for plants.

Screening: blue-white colony assay (lacZ α-complementation), antibiotic selection, colony hybridisation, PCR, sequencing.

PCR cycle: denaturation 94-95 °C, annealing 50-65 °C, extension 72 °C (Taq); 2^n amplification; primers must be specific and 18-25 nt.

CRISPR-Cas9 uses guide RNA to direct Cas9 double-strand cut; PAM requirement (NGG for SpCas9); repair by NHEJ (knockout) or HDR (knock-in).

Murashige and Skoog (MS) medium is the standard; components: macroelements, microelements, vitamins, sucrose (2-3%), plant growth regulators, agar (0.8%) at pH 5.7-5.8.

Auxin/cytokinin ratio rule: high auxin promotes root, high cytokinin promotes shoot, intermediate ratio gives callus.

Culture types: callus, suspension, protoplast, anther (haploid production), embryo, meristem (virus-free plants), somatic embryogenesis.

Totipotency underlies regeneration; hardening of in vitro plantlets is required before field transfer.

Micropropagation stages: initiation, multiplication, rooting, hardening; somaclonal variation is a source of useful variants.

Agrobacterium tumefaciens transfers T-DNA from Ti plasmid; vir genes induced by acetosyringone; binary vector systems (e.g., pBI121).

Bt cotton expresses Cry1Ac from Bacillus thuringiensis; Golden Rice produces beta-carotene; RNAi used for virus resistance.

Culture types: primary (finite lifespan), secondary, continuous cell lines (HeLa, CHO, HEK293); anchorage-dependent cells need surface, suspension cells do not.

Medium: DMEM/RPMI-1640 with 5-10% fetal bovine serum, antibiotics, glutamine; serum-free media reduce variability and contamination risk.

Incubator conditions: 37 °C, 5% CO2 for bicarbonate buffering, humidified; aseptic laminar flow hood (class II) for handling.

Subculturing uses trypsin-EDTA to detach adherent cells; split ratio and confluency determine schedule; cryopreservation in 10% DMSO at -196 °C.

Hybridoma technology (Köhler and Milstein, 1975): B cell fused with myeloma gives monoclonal antibodies.

Transgenic animals: pronuclear microinjection, somatic cell nuclear transfer (Dolly, 1996), lentiviral vectors; applications in biopharming and disease models.

Stem cells: embryonic (pluripotent) vs adult (multipotent); induced pluripotent stem cells (Yamanaka factors Oct4, Sox2, Klf4, c-Myc).

Fermenter types: stirred tank (STR), airlift, bubble column, packed bed; STR has impeller for mixing and baffles to prevent vortex.

Key parameters: aeration (vvm), agitation (rpm), temperature, pH, dissolved oxygen, foam; kLa measures oxygen transfer capacity.

Sterilisation of fermenter by steam-in-place; media sterilised by continuous or batch heat treatment.

Fermentation products: ethanol (Saccharomyces cerevisiae), citric acid (Aspergillus niger), penicillin (Penicillium chrysogenum), glutamic acid (Corynebacterium glutamicum), vitamin B12 (Pseudomonas denitrificans).

Downstream processing sequence: cell disruption, solid-liquid separation (centrifugation, filtration), concentration (ultrafiltration, evaporation), purification (chromatography), formulation.

Immobilised enzymes/cells improve stability and allow continuous operation; examples: glucose isomerase for high-fructose syrup, penicillin acylase.

Scale-up criteria: constant tip speed, constant kLa, constant power per unit volume; choice depends on oxygen demand and shear sensitivity.

BOD5 measures oxygen consumed by microbes degrading organic matter in 5 days at 20 °C; COD measures chemical oxidation, COD ≥ BOD.

Activated sludge process: aeration tank + secondary clarifier; parameters F/M ratio, sludge age (SRT), mixed liquor suspended solids (MLSS).

Indicator organisms: E. coli and coliforms indicate faecal contamination; MPN test and membrane filtration for potable water.

Bioremediation strategies: in situ (bioventing, bioaugmentation) vs ex situ (land farming, composting, slurry reactors); monitored natural attenuation.

Phytoremediation categories: phytoextraction, phytostabilisation, rhizofiltration, phytovolatilisation; hyperaccumulators (e.g., Brassica juncea for metals).

Anaerobic digestion stages: hydrolysis, acidogenesis, acetogenesis, methanogenesis; biogas ~60% methane, 40% CO2.

Oil spill response: biosurfactants, oil-zapper (Pseudomonas putida with xylene monooxygenase), nutrient addition (biostimulation).

Recombinant human insulin (Humulin, 1982) was first approved rDNA drug; produced in E. coli or yeast.

Monoclonal antibodies: suffix -mab; humanised (-zumab), chimeric (-ximab), fully human (-mumab); used in cancer, autoimmune disease.

Vaccine platforms: live attenuated, inactivated, subunit, toxoid, conjugate, mRNA (lipid nanoparticle), viral vector; cold chain requirements differ.

Gene therapy vectors: AAV (non-integrating), lentivirus (integrating), adenovirus (transient); approved examples include CAR-T (Kymriah).

Pharmacokinetics: ADME; bioavailability, half-life, therapeutic index = TD50/ED50.

Diagnostics: ELISA, PCR/RT-PCR, lateral flow immunoassay, next-generation sequencing; biomarker validation requires sensitivity and specificity.

Biosimilars are follow-on biologics approved after patent expiry; comparability exercises demonstrate similarity to reference product.

Innate immunity: physical barriers, phagocytes, complement, pattern recognition receptors (TLR); adaptive immunity: B cells (antibody) and T cells (cell-mediated).

Antibody structure: two heavy and two light chains, Fab (antigen binding) and Fc (effector); isotypes IgG, IgM, IgA, IgE, IgD.

IgM is first responder and pentamer; IgG is most abundant and crosses placenta; IgA is mucosal dimer; IgE mediates allergy.

MHC class I presents endogenous peptides to CD8+ cytotoxic T cells; MHC class II presents exogenous peptides to CD4+ helper T cells.

Primary response has lag and low IgM→IgG switch; secondary response is faster, larger, higher affinity (memory cells).

Vaccine types: live attenuated (BCG, OPV), killed (IPV), toxoid (tetanus), subunit (HBsAg), conjugate (Hib), mRNA.

Herd immunity threshold depends on R0: threshold = 1 - 1/R0; higher R0 requires higher coverage.

Hypersensitivity types I-IV: immediate IgE, antibody-mediated, immune complex, delayed T-cell (tuberculin test).

Cartagena Protocol on Biosafety regulates transboundary movement of living modified organisms; India is a party; Nagoya Protocol covers access and benefit sharing.

Indian regulatory chain: Institutional Biosafety Committee → Review Committee on Genetic Manipulation (RCGM, DBT) → Genetic Engineering Appraisal Committee (GEAC, MoEFCC) for environmental release.

Patents: novelty, inventive step, industrial application; Indian Patents Act Section 3(j) excludes plants and animals; microorganisms and essentially biological processes are excluded, but microbiological processes are patentable.

TRIPS requires 20-year patent term; India allows product patents in pharmaceuticals since 2005; compulsory licensing provision (Section 84).

GEAC approval required for field trials; Bt cotton is the only approved GM crop for commercial cultivation in India.

Biosafety levels: BSL-1 (low risk) to BSL-4 (dangerous pathogens); containment, PPE, HEPA filtration, autoclave waste.

Odisha applications: rice and pulse improvement at OUAT and NRRI (Cuttack); ILS Bhubaneswar works on infectious disease and immunology; marine biotechnology at Berhampur University; DBT-OUAT collaboration on crop biotechnology.

Biological Diversity Act 2002 and Odisha Biodiversity Board govern access to local biological resources; prior informed consent needed for commercial use.