BSEH SS Biotechnology cheat sheet

Biotechnology = use of living organisms, cells, or their components (enzymes, DNA) to make products or provide services; the OECD definition emphasizes industrial application of biological systems.

Classical (old) biotechnology: fermentation-based products such as bread, beer, wine, cheese, vinegar, and antibiotics like penicillin; it does not require recombinant DNA.

Modern biotechnology relies on recombinant DNA, gene cloning, monoclonal antibodies, and tissue culture; it began with Cohen-Boyer (1973) and the first recombinant human insulin (1982).

Colour code: red = medical/pharmaceutical, green = agricultural, white/grey = industrial and environmental, blue = marine/aquatic, yellow = food biotechnology.

Scope spans health (vaccines, insulin, gene therapy), agriculture (Bt crops, biofertilizers), industry (enzymes, biofuel), environment (bioremediation), and forensics (DNA fingerprinting).

Biotechnology is an interdisciplinary field combining microbiology, biochemistry, genetics, molecular biology, chemical engineering, and bioinformatics.

Bioinformatics applies computation to biological data (sequence alignment, BLAST, genome annotation); it is the data-handling arm of modern biotechnology.

Milestones: 1953 DNA double helix, 1973 recombinant DNA, 1977 Sanger sequencing, 1983 PCR, 1990 Human Genome Project start, 2003 human genome draft completed.

Scale-up distinguishes biotechnology from pure biology: a lab-scale process must be translated to bioreactors under controlled pH, temperature, aeration, and sterility.

Prokaryotic cell: no membrane-bound nucleus or organelles, 70S ribosomes (50S + 30S), single circular chromosome, peptidoglycan cell wall in bacteria.

Eukaryotic cell: true nucleus, 80S ribosomes (60S + 40S), membrane-bound organelles; mitochondria and chloroplasts carry their own circular DNA (endosymbiotic theory).

Membrane model = fluid mosaic: phospholipid bilayer with integral and peripheral proteins, cholesterol modulating fluidity; transport may be passive, facilitated, or active (ATP-driven).

Protein structure levels: primary (amino acid sequence), secondary (alpha helix, beta sheet), tertiary (3D folding), quaternary (multiple polypeptide subunits).

Enzymes lower activation energy; Michaelis-Menten: Km = substrate concentration at half Vmax (inverse measure of affinity), Vmax reflects enzyme amount/turnover.

Nucleic acids: DNA is double-stranded, deoxyribose, bases A-T and G-C; RNA is usually single-stranded, ribose, A-U and G-C; phosphodiester bonds link nucleotides 5' to 3'.

Carbohydrates: monosaccharides (glucose, fructose), disaccharides (sucrose, lactose), polysaccharides (starch, glycogen, cellulose, chitin); cellulose and chitin are structural.

Lipids: triglycerides (energy storage), phospholipids (membranes), steroids (cholesterol, hormones); amphipathic nature drives bilayer formation.

Cell cycle: G1, S (DNA replication), G2, M; checkpoints at G1/S and G2/M; apoptosis is programmed cell death distinct from necrosis.

Central dogma: DNA to RNA (transcription) to protein (translation); retroviruses reverse this with reverse transcriptase (RNA to DNA).

Semiconservative replication: helicase unwinds, primase makes RNA primer, DNA polymerase III synthesizes 5' to 3', ligase seals Okazaki fragments.

Mendelian ratios: monohybrid F2 = 3:1 phenotype, 1:2:1 genotype; dihybrid F2 = 9:3:3:1 when genes are unlinked and assort independently.

Codominance (AB blood group) vs incomplete dominance (pink snapdragon from red x white); multiple alleles exist at a locus though a diploid carries two.

Mutations: point substitutions (missense, nonsense, silent), frameshift insertions/deletions, chromosomal aberrations (deletion, duplication, inversion, translocation).

Lac operon is negatively controlled: lactose (as allolactose) inactivates the repressor, enabling transcription; glucose presence causes catabolite repression via low cAMP.

Genetic code is degenerate, non-overlapping, triplet, and nearly universal; 64 codons code 20 amino acids plus start (AUG) and three stop codons (UAA, UAG, UGA).

Hardy-Weinberg equilibrium: p + q = 1 and p^2 + 2pq + q^2 = 1; deviations indicate selection, mutation, drift, migration, or non-random mating.

Epistasis, pleiotropy, and polygenic inheritance explain deviations from simple Mendelian ratios; sex-linked traits show different frequencies in males and females.

Restriction endonucleases cut specific palindromic sequences (EcoRI: GAATTC, sticky ends; SmaI: CCCGGG, blunt ends); sticky ends ligate more efficiently.

Cloning workflow: isolate DNA, cut vector and insert with same enzyme, ligate, transform host, select (antibiotic resistance), screen (blue-white, colony hybridization).

Plasmids are extrachromosomal circular vectors with origin of replication, selectable marker, and multiple cloning site; pBR322 and pUC19 are classic examples.

Transformation methods: CaCl2 heat shock, electroporation, microinjection, gene gun (biolistics), and Agrobacterium tumefaciens Ti plasmid for plants.

PCR cycle: denaturation (~94 C), annealing (~50-65 C, primer Tm dependent), extension (~72 C by Taq polymerase); n cycles give roughly 2^n-fold amplification.

CRISPR-Cas9 uses a guide RNA to direct Cas9 nuclease to a target sequence adjacent to a PAM; it creates a double-strand break for knockout or repair-mediated editing.

DNA libraries: genomic library contains all sequences; cDNA library contains only expressed genes and lacks introns, made from mRNA via reverse transcriptase.

Southern blot detects DNA, Northern detects RNA, Western detects protein; each uses probe or antibody after gel separation and transfer.

Sanger sequencing uses dideoxynucleotides (chain terminators); next-generation sequencing uses massively parallel short reads and bioinformatic assembly.

Bacterial growth curve phases: lag, log (exponential), stationary, decline; generation time varies from ~20 min (E. coli) to hours for slow growers.

Sterilization kills all microbes including spores (autoclave 121 C, 15 psi, 15-20 min); disinfection reduces pathogens on surfaces; pasteurization reduces spoilage organisms in food.

Gram stain: crystal violet, iodine, alcohol decolourization, safranin counterstain; Gram-positive retains purple due to thick peptidoglycan, Gram-negative appears pink.

Culture media: selective (inhibits unwanted microbes, e.g., MacConkey for Gram-negative), differential (distinguishes colonies, e.g., blood agar), enrichment (favours target organism).

Industrial fermentation: batch (closed, fixed nutrients), fed-batch (nutrients added), continuous (steady state, chemostat); stirred-tank bioreactors control pH, DO, temperature.

Key microbial products: penicillin (Penicillium), citric acid (Aspergillus niger), ethanol (Saccharomyces cerevisiae), insulin (recombinant E. coli), streptomycin (Streptomyces).

Nitrogen fixation by Rhizobium (symbiotic) and Azotobacter (free-living); nitrification by Nitrosomonas and Nitrobacter; denitrification returns nitrogen to atmosphere.

Bioremediation uses microbes to degrade pollutants (oil spills, pesticides); biosorption and bioleaching apply microbes to metal recovery.

Viruses are obligate intracellular parasites with nucleic acid and capsid; bacteriophages infect bacteria and are used in cloning and phage display.

Totipotency is the capacity of a plant cell to regenerate a whole plant; it underpins micropropagation.

Callus induction requires auxin-to-cytokinin balance: high auxin favours roots, high cytokinin favours shoots, intermediate favours callus.

Micropropagation stages: explant selection, surface sterilization, callus/shoot initiation, rooting, hardening, field transfer; produces clonal disease-free plants.

Meristem culture yields virus-free plants because the apical meristem divides faster than virus spread; thermotherapy can be combined.

Somatic embryogenesis (embryo-like structures) and protoplast fusion (somatic hybridization) create hybrids; anther culture produces haploid plants for breeding.

Animal cell culture: primary cultures have limited lifespan; continuous cell lines (HeLa, CHO) are immortal; media need serum or defined supplements, 37 C, 5% CO2.

Trypsinization subcultures adherent cells; suspension culture suits hybridomas and vaccine production; cryopreservation in liquid nitrogen preserves stocks.

Monoclonal antibodies from hybridoma technology (Kohler and Milstein) fuse B cells with myeloma cells for indefinite antibody production.

Contamination control is critical: laminar flow hoods, HEPA filtration, autoclaved glassware, and aseptic technique prevent fungal and bacterial contamination.

Innate immunity is non-specific and immediate (skin, phagocytes, complement, inflammation); adaptive immunity is specific and has memory (B and T lymphocytes).

Antibody structure: two heavy and two light chains, Y-shaped, with variable regions forming the antigen-binding site and constant regions determining isotype (IgG, IgM, IgA, IgE, IgD).

IgM is the first antibody in primary response; IgG is most abundant and crosses the placenta; IgA is secretory (mucosal); IgE mediates allergy.

Vaccines: live attenuated (BCG, MMR), inactivated (polio Salk), toxoid (tetanus), subunit/conjugate (HBV, Hib), mRNA (COVID-19).

ELISA formats: direct, indirect (detects patient antibody), sandwich (detects antigen), competitive; enzyme label converts substrate to a colour signal read spectrophotometrically.

Rapid diagnostic tests use lateral flow with colloidal gold-labelled antibodies; PCR-based tests detect pathogen nucleic acid with high sensitivity.

Hypersensitivity types: I (IgE, anaphylaxis), II (antibody-mediated, transfusion reactions), III (immune complex, Arthus), IV (delayed, T-cell, TB skin test).

Blood grouping uses ABO and Rh antigens; agglutination indicates the presence of corresponding antigen on red cells.

Autoimmune disease arises from loss of self-tolerance (type 1 diabetes, rheumatoid arthritis); immunodeficiency may be primary (genetic) or secondary (HIV).

Bt crops carry Bacillus thuringiensis cry genes producing insecticidal proteins toxic to specific larvae but not mammals.

Golden rice is engineered to synthesize beta-carotene (provitamin A) to combat deficiency; biofortification enriches staple crops with nutrients.

Transgenic animals serve as bioreactors (pharming, e.g., milk-borne therapeutic proteins), disease models, and organ donors in xenotransplantation research.

Recombinant human insulin (Humulin) from E. coli or yeast replaced animal-derived insulin; recombinant factor VIII, erythropoietin, and growth hormone are similar products.

Gene therapy delivers corrective genes via viral vectors (adeno-associated, lentivirus) or non-viral methods; somatic edits are not inherited, germline edits are heritable.

Industrial enzymes: amylase (starch processing), protease (detergents), lipase (food), cellulase (biofuel); immobilized enzymes allow reuse and continuous operation.

Biofuels: bioethanol from sugar/starch fermentation, biodiesel from transesterified oils, biogas (methane) from anaerobic digestion.

Single-cell protein and fermentation-derived foods (mycoprotein, amino acids, vitamins) are industrial fermentation products.

Molecular diagnostics and pharmacogenomics tailor drug choice to genotype, e.g., HER2 testing before trastuzumab therapy.

Biosafety levels: BSL-1 low risk, BSL-2 moderate, BSL-3 serious/lethal agents, BSL-4 dangerous/exotic agents; controls increase with level (PPE, containment, airflow).

Cartagena Protocol on Biosafety governs transboundary movement of living modified organisms; the precautionary principle guides risk decisions.

Informed consent, confidentiality, and equitable benefit-sharing are core bioethics principles; the Nuremberg Code and Declaration of Helsinki set research ethics standards.

Ethical issues: germline gene editing, human cloning, genetic discrimination, patenting of genes, animal welfare in research (3Rs: replacement, reduction, refinement).

Intellectual property: patents protect inventions (novel, non-obvious, industrially applicable); plant varieties may be protected by plant breeders' rights or sui generis systems.

TRIPS Agreement requires member states to provide patent protection for microorganisms and microbiological processes, but allows exclusion of plants and animals.

Biological Diversity Act and similar national laws regulate access to biological resources and benefit-sharing; biopiracy is unauthorized exploitation of genetic resources.

Biosafety practices: autoclave infectious waste, disinfect spills, use biosafety cabinets for aerosols, restrict access, and report incidents.

Ethical review boards (IRB/IACUC) approve human and animal research; data integrity and authorship criteria (substantial contribution) are part of research ethics.

Micropipettes: choose the smallest volume range that fits the volume; never exceed the stated maximum; pre-wet tips for accurate delivery of viscous liquids.

Serial dilution: each step reduces concentration by a fixed factor; 10-fold serial dilutions give 1/10, 1/100, 1/1000 of the original.

Centrifugation separates by density and size; relative centrifugal force depends on rotor radius and speed; balance tubes and use appropriate g-force and time.

Spectrophotometry: Beer-Lambert law A = epsilon x c x l; DNA concentration estimated from A260 (A260 of 1 = ~50 ng/microlitre for dsDNA).

Agarose gel electrophoresis separates DNA by size; DNA migrates toward the anode (positive electrode) because it is negatively charged; smaller fragments move faster.

PCR setup: keep reagents on ice, use sterile filter tips, include negative control (no template) and positive control; contamination causes false positives.

Chromatography: gel filtration separates by size, ion exchange by charge, affinity by specific binding; used for protein purification.

Good laboratory practice: label reagents with date and concentration, record raw data in a bound notebook, calibrate instruments, dispose of waste by category.

Microscopy: adjust condenser and iris for contrast; oil immersion (100x objective) increases resolution; calculate total magnification as objective x eyepiece.