Unit 4: Biotechnology

Chapter 1: Principles and Processes

Principles of Biotechnology

1. Genetic Engineering:
   • Altering the chemistry of DNA and RNA.
   • Introducing it into host organisms to change their phenotype.
2. Bioprocess Engineering:
   • Maintaining a sterile environment for the growth of desired microbes.
   • Enables large-scale production of products like antibiotics, vaccines, etc.

Tools of Recombinant DNA Technology

• Restriction Enzymes: Molecular scissors to cut DNA at specific sites.
• Cloning Vector: A DNA molecule (e.g., plasmid) that carries foreign DNA into a host.
• Ligase: Enzyme that joins DNA fragments.
• Host Organism: Usually bacteria (E. coli) or yeast.

Steps in Recombinant DNA Technology

1. Isolate DNA (from source and vector).
2. Cut both DNAs with the same restriction enzyme.
3. Amplify the gene of interest using PCR.
4. Ligate the gene into the vector to create rDNA.
5. Transform the host cell with the rDNA.
6. Select and screen for transformed cells.
7. Culture the cells to obtain the foreign gene product.

Polymerase Chain Reaction (PCR)

• A technique to amplify a specific DNA segment in vitro.
• Steps in one cycle:
  1. Denaturation: Heat to separate DNA strands (~94°C).
  2. Annealing: Primers bind to template DNA (~54°C).
  3. Extension: Taq polymerase synthesizes new DNA (~72°C).
• Results in exponential amplification.

Gene Transfer Methods

• Transformation (Heat Shock): Making bacterial cells competent to take up DNA.
• Microinjection: Directly injecting DNA into an animal cell nucleus.
• Biolistics (Gene Gun): Bombarding plant cells with DNA-coated microparticles.
• Disarmed Pathogen Vectors: Using modified viruses or bacteria to deliver genes.

Selection of Recombinants

• Antibiotic Resistance:
  • If a resistance gene in the vector is disrupted by the insert, the cell will lose that resistance.
• Blue-White Screening (Insertional Inactivation):
  • Recombinants (with insert in lacZ gene) are white.
  • Non-recombinants (intact lacZ gene) are blue.

Bioreactors

• Large vessels for large-scale culture of cells to produce biological products.
• Provide optimal conditions (temperature, pH, oxygen, etc.).
• Types:
  • Stirred-Tank Bioreactor
  • Sparged Stirred-Tank Bioreactor

Downstream Processing

• The final stage of production.
• Involves:
  • Separation of the product from the culture.
  • Purification of the product.
  • Formulation with preservatives.
  • Quality control and clinical trials.