Biotechnology: Principles and Processes

• Introduction

  • Definition: Using organisms or enzymes to produce products for humans
  • EFB Definition: Integration of natural science and organisms for products and services
  • Core Techniques
    • Genetic Engineering: Altering DNA/RNA chemistry
    • Bioprocess Engineering: Maintaining sterile conditions for microbial growth

• Principles of Genetic Engineering

  • Creation of Recombinant DNA (rDNA)
  • Gene cloning and gene transfer
  • Concept of Origin of Replication (ori)
  • Prevention of foreign DNA integration without ori
  • First recombinant DNA construction (Stanley Cohen and Herbert Boyer, 1972)
    • Antibiotic resistance gene linked to plasmid vector

• Tools of Recombinant DNA Technology

  • Restriction Enzymes (Molecular Scissors)
    • Types: Exonucleases (remove nucleotides from ends) and Endonucleases (cut at specific sites)
    • Recognition Sequences: Palindromic nucleotide sequences
    • Action: Creates sticky ends for easy joining
  • Cloning Vectors (Vehicles for Gene Transfer)
    • Features required: Ori site, Selectable marker, Cloning sites
    • Examples: Plasmids, Bacteriophages
    • Vectors for plants/animals: Agrobacterium tumefaciens (plants), Retroviruses (animals)
  • Competent Host (For Transformation)
    • Methods to make host take up rDNA:
      • Chemical treatment (Divalent cations like $Ca^{2+}$)
      • Heat shock method ($42^\circ C$)
      • Microinjection
      • Biolistics / Gene gun (gold/tungsten particles)
      • Disarmed pathogen vectors

• Processes of Recombinant DNA Technology

  • Isolation of Genetic Material (DNA)
    • Cell lysis using enzymes (Lysozyme, Cellulase, Chitinase)
    • Precipitation using chilled ethanol
  • Cutting of DNA at Specific Locations
    • Restriction enzyme digestion
    • Agarose Gel Electrophoresis
      • Separation based on size
      • Staining with Ethidium Bromide
      • Visualization under UV light
      • Elution: Extraction of DNA bands from gel
  • Amplification of Gene of Interest
    • Polymerase Chain Reaction (PCR)
    • Three steps per cycle:
      1. Denaturation (High temperature)
      2. Annealing (Primer binding)
      3. Extension (Taq polymerase action)
    • Uses thermostable Taq polymerase
  • Insertion of rDNA into Host Cell/Organism
    • Direct introduction of recombinant DNA into recipient host
  • Culturing Host Cells on a Large Scale
    • Bioreactors (Vessels for large-volume processing)
    • Types:
      • Simple stirred-tank bioreactor
      • Sparged stirred-tank bioreactor
    • Parameters controlled: Temperature, pH, oxygen, foam, vitamins
  • Downstream Processing
    • Separation and purification of products
    • Formulation with preservatives
    • Strict quality control testing