Mutations, Gene Transfer & Genetic Engineering

Mutant Isolation via Plating

Positive selection: stamp master plate onto nutrient plate ± antibiotic; colonies that grow on antibiotic plate = resistant mutants.
Negative selection: duplicate onto plates ± \text{histidine}; colonies that grow only on supplemented plate = his^- auxotrophs (nutritional mutants).

Start with his^- auxotroph of Salmonella.
Incubate with suspected mutagen + rat‐liver extract ➔ plate on medium lacking histidine.
Revertant colonies (regained his^+) indicate mutagenicity ≈ potential carcinogen.

Three mechanisms transfer DNA between bacteria (no reproduction involved).
• Transformation: uptake of naked DNA.
• Conjugation: plasmid copy passed via type-IV pilus/mating bridge.
• Transduction: DNA moved by bacteriophage.

DNA fragment must find homologous region; otherwise degraded.
Plasmid uptake bypasses homologous requirement → higher success.
Competency varies: e.g., E.\ coli tries any DNA; S.\ pneumoniae prefers own species.

Plasmid-only process; requires fertility (F) factor.
Gram-negatives: pilus can bridge short distance; Gram-positives: pilus draws cells into direct contact (mating bridge).
Spreads antibiotic resistance rapidly (colony-wide in 2–4 h).
Plasmid cargo worth knowing: antibiotic resistance, antibiotic synthesis, virulence genes, toxins.
Plasmid incompatibility: competing plasmids with same promoter ➔ only tighter-binding expressed (therapeutic strategy to silence resistance).

Generalized (lytic phage): random host DNA mistakenly packaged; requires homologous recombination → usually fails.
Specialized (temperate/lysogenic phage): prophage excision carries specific flanking host genes; phage integrase enables insertion → high success; major source of bacterial virulence factors (e.g., diphtheria toxin, EHEC Shiga toxin).

PCR amplify gene of interest (GOI) with sticky ends; cycles = denature → anneal → extend using heat-stable polymerase (e.g., Taq).
Cut plasmid & GOI with same restriction enzyme (e.g., EcoRI: \text{GAATTC} palindromic site).
Ligate to form recombinant plasmid.
Transform bacteria.
Screen colonies:
• Antibiotic plate selects cells that received any plasmid.
• Blue/white screening: lacZ in multiple-cloning site—interrupted \Rightarrow white (recombinant); intact \Rightarrow blue (native plasmid).

Agrobacterium\ tumefaciens TI plasmid drives inserted gene into plant chromosome.
Uses: stable transgenic crops (drought, pest, heat resistance) or transient expression bioreactors (vacuum-infiltrate wounded leaves; short-term overproduction of therapeutics, then harvest & discard plant).

Nucleic acids: gene therapy (e.g., sickle-cell cure), diagnostic probes.
Recombinant microbes: industrial enzymes, vaccines, insulin, bioremediation.
Transgenic plants: stress-tolerant foods, higher yield cotton, plant-made vaccines/ drugs.
Transgenic animals:
• AquAdvantage salmon (fast-growing).
• Humanized mice (Regeneron) for drug testing.
• Xenografting: editing pigs to provide human-compatible organs.