Microbial Genetics & Recombinant DNA – Exam Review

Replica Plating Techniques

  • Master plate stamped onto two media via sterile velvet
  • Antibiotic-supplemented plate → colonies that grow are antibiotic-resistant mutants
  • Histidine-supplemented vs. histidine-lacking plates → growth only on supplemented plate identifies a histidine auxotroph (nutritional mutant)

Ames Test (Mutagen/Carcinogen Screen)

  • Uses a histidine auxotroph of Salmonella
  • Mix: auxotroph + suspected mutagen + rat-liver extract
  • Plate on medium lacking histidine
  • Revertant colonies (His⁺ prototrophs) indicate mutagenic potential; ↑ revertants ⇒ suspected carcinogen

Horizontal Gene Transfer (HGT)

  • HGT introduces foreign DNA without reproduction; 33 major mechanisms:
    • Transformation: uptake of naked DNA; success depends on cell competence & homologous recombination (chromosomal) or plasmid uptake
    • Conjugation: plasmid copy transferred through type IV pilus/mating bridge; key driver of antibiotic resistance
    • Transduction: bacteriophage-mediated DNA transfer
      • Generalized (lytic phage packages random host DNA)
      • Specialized (lysogenic phage excises with adjacent host genes via integrase)

Plasmid Basics

  • Extrachromosomal, self-replicating DNA; often carry fertility genes (pilus), promoters & selectable traits
  • Clinically relevant genes: antibiotic resistance, antibiotic synthesis, virulence factors, toxin genes
  • Plasmid incompatibility: competing plasmids with same promoter; one silences the other ⇒ potential therapeutic approach to suppress resistance

Transformation in the Lab (Core Genetic-Engineering Workflow)

  • Amplify gene of interest (GOI) via PCR (heat-stable polymerase e.g., Taq)
  • Cut GOI & cloning vector with same restriction enzyme (e.g., EcoRI) → compatible sticky ends
  • Ligate to form recombinant plasmid
  • Introduce plasmid into competent bacteria (chemical or electroporation transformation)

Selection & Screening

  • Plate on medium containing antibiotic → only cells with any plasmid survive
  • Blue-white screen (lacZ disruption)
    • Blue colonies: non-recombinant plasmid (lacZ intact)
    • White colonies: recombinant plasmid (lacZ disrupted by GOI)

Plant Genetic Engineering (Ti Plasmid)

  • Agrobacterium tumefaciens Ti plasmid delivers insert into plant chromosome
  • Stable integration (crop improvement) or transient expression (rapid protein production)
  • Traits engineered: stress tolerance (drought/heat), pest resistance, yield or nutrient enhancement, therapeutic protein production

Key Applications of Recombinant DNA

  • Gene therapy (e.g., sickle-cell disease), molecular diagnostics
  • Recombinant microbes: industrial enzymes, vaccines, biopharmaceuticals (insulin), bioremediation
  • Transgenic plants: hardier food crops, edible vaccines, specialty metabolites
  • Transgenic animals:
    • Aquaculture (rapid-growing salmon)
    • Pharming (therapeutic proteins in milk/eggs)
    • Humanized disease models (immune-system or organ replacement genes)
    • Xenografting: gene-edited pig organs with human antigens for transplantation