A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques, introducing, modifying, or removing specific genes. The first GMO was a bacterium created by Herbert Boyer and Stanley Cohen in 1973 by inserting a frog gene into E. coli. The first GMO crop approved for commercial cultivation was Bt cotton in India (2002) and Flavr Savr tomato in the USA (1994). GMO technology has applications in agriculture (insect-resistant, herbicide-tolerant, nutritionally enhanced crops), medicine (insulin, growth hormone, vaccines, clot-busting drugs produced in bacteria/yeast), environmental bioremediation, and industrial biotechnology. The creation of GMOs involves recombinant DNA technology: restriction enzymes, vectors (plasmids, viruses), host organisms, and selection systems.

Bt cotton (Bacillus thuringiensis cotton) is a genetically modified cotton variety containing the cry gene (crystal protein gene) from the soil bacterium Bacillus thuringiensis. The cry gene encodes Bt toxin (Cry proteins = δ-endotoxins), a protein that is toxic to certain insects — particularly lepidopteran larvae (cotton bollworm, Helicoverpa armigera). Mechanism: Bt toxin ingested by insect larvae → in alkaline insect midgut, protoxin is activated to toxin → binds to specific receptors on midgut cells → forms pores in cell membrane → cell lysis → larva dies. Non-toxic to mammals (requires alkaline gut + specific receptors). Approved in India in 2002. Bt cotton reduces insecticide use by ~50% → economic and environmental benefit. Resistance concern: some populations of bollworm developing resistance → refuge strategy (planting non-Bt cotton around Bt fields to maintain susceptible population). Other Bt crops: Bt brinjal (Bt eggplant) — approved in Bangladesh, under development in India.

Thermus aquaticus is a thermophilic bacterium (thrives at high temperatures) discovered by Thomas Brock in hot springs at Yellowstone National Park (1969), where temperatures reach 70-80°C. T. aquaticus provided Taq DNA polymerase — a thermostable DNA polymerase that can withstand the high temperatures needed in PCR (Polymerase Chain Reaction). Critical properties of Taq polymerase: Thermostability: active up to 95°C, optimally at 72°C. Allows repeated denaturation steps (94°C) in PCR without enzyme degradation. Before Taq, PCR required adding fresh enzyme after each heating step. High fidelity (relative to early PCR standards, though not as accurate as Pfu polymerase from Pyrococcus furiosus). Processivity: can synthesise several hundred to 1000+ base pairs per cycle. Error rate: ~1 error per 10⁵ bases. Lacks 3'→5' proofreading exonuclease activity (some other thermostable polymerases have this). Discovery of Taq polymerase by Kary Mullis and its application in PCR earned Mullis the Nobel Prize in Chemistry in 1993.

Agrobacterium tumefaciens is a soil bacterium that naturally causes crown gall disease in plants. It is the most widely used vector system for plant transformation. The Ti plasmid (Tumour-inducing plasmid) is the natural genetic tool: T-DNA (Transferred DNA): a specific segment of Ti plasmid that is naturally transferred from the bacterium to plant cells and stably integrated into the plant nuclear genome. T-DNA contains genes for: (1) Phytohormone synthesis (auxins + cytokinins) → causes uncontrolled cell proliferation (tumour/crown gall). (2) Opine synthesis genes (nopaline, octopine) → produce opines (unusual amino acid derivatives) that only the bacterium can use as food. For plant transformation: the tumour-causing genes are REMOVED from T-DNA (disarmed) and replaced with the gene of interest. The vir (virulence) genes on Ti plasmid remain — they are responsible for T-DNA transfer. The disarmed T-DNA containing the gene of interest is then delivered into plant cells. Transformed plant cells regenerated into whole plants via tissue culture.

pBR322 is one of the most widely used early cloning vectors, derived from Escherichia coli plasmids. It was constructed by Bolivar and Rodriguez (BR = their initials, 322 = clone number) in 1977. Properties: Small plasmid (4361 bp). Circular double-stranded DNA. Contains: origin of replication (ori) — allows autonomous replication in E. coli. Two antibiotic resistance genes: ampicillin resistance (Amp^R) and tetracycline resistance (Tet^R) — used for selection. Multiple restriction enzyme sites (BamHI in Tet^R gene, SalI in Tet^R gene, PstI in Amp^R gene). Insertional inactivation: when foreign DNA inserted at BamHI site → disrupts Tet^R gene → bacteria sensitive to tetracycline but resistant to ampicillin → easy identification. pBR322 is maintained in E. coli (gram-negative bacterium) and replicates to high copy number (~15-20 copies/cell). It served as the foundation for many subsequent cloning vectors (pUC, pBluescript, pET series).

PCR amplifies specific DNA sequences exponentially. Three steps in each cycle: (1) Denaturation: 94-95°C for 30-60 seconds → hydrogen bonds between DNA strands broken → single-stranded template. (2) Annealing: 50-65°C for 30-60 seconds → short synthetic oligonucleotide primers (18-25 bp) bind to complementary sequences on template strands. Primer design is critical for specificity. (3) Extension: 72°C for 30-60 seconds → Taq polymerase synthesises new DNA strand from primer, reading template 3'→5', synthesising 5'→3'. One cycle → each original molecule becomes 2. After n cycles: 2ⁿ copies. 30 cycles → ~10⁹ copies (from 1 molecule). Applications: DNA forensics, prenatal diagnosis of genetic disorders, detection of pathogens (COVID-19 RT-PCR), cloning, site-directed mutagenesis, gene expression analysis (RT-PCR, qPCR), ancient DNA analysis, cancer mutation detection.

Transgenic animals contain foreign genes (transgenes) introduced into their genome. Methods: pronuclear microinjection (inject DNA into fertilised egg pronucleus), retroviral infection, embryonic stem (ES) cell technology. Rosie the cow (1997): produced human protein-enriched milk (with α-lactalbumin → better nutrition for babies). Transgenic mice: most widely used model organisms for studying gene function, disease mechanisms, drug testing. 'Oncomouse'/'Harvard mouse': first patented transgenic animal, carries human cancer gene (ras oncogene) → prone to cancer → used in cancer research. Dolly the sheep (1997): first mammal cloned from adult somatic cell (mammary gland cell of a Finn Dorset sheep + enucleated egg from Scottish Blackface sheep → surrogate Blackface sheep). Proved somatic cell nuclear transfer (SCNT) possible. Production of pharmaceutical proteins in transgenic animals (pharming): Factor VIII (blood clotting), tPA (tissue plasminogen activator) in sheep milk.

The Human Genome Project (HGP): started 1990, completed 2003. International effort by 20 institutions in 6 countries. Goal: determine the complete sequence of all 3 billion base pairs of the human genome and identify all human genes. Key findings: ~20,000-25,000 protein-coding genes (less than expected). ~1.5% of genome codes for proteins. ~50% of genome consists of repetitive sequences. >99.9% of base sequence identical between humans. Used Sanger sequencing (chain termination) and shotgun sequencing. Application: understanding genetic basis of diseases, developing new drugs, personalised medicine, gene therapy, forensics. Draft sequence released 2001. Complete sequence 2003. Model organisms sequenced: E. coli (1997), yeast (1996), C. elegans (1998), Arabidopsis thaliana (first plant, 2000), rice genome (2002-2005). Bioinformatics: computational analysis of biological sequence data — BLAST, genome annotation, protein structure prediction (AlphaFold).