22: Biotechnology & Molecular Cloning

Difference between cellular and molecular biotechnology?

• Cellular biotechnology: Uses whole organisms directly.
  

  • Examples: Fermented fish storage (cold/anaerobic → preservation + flavor), bread/beer (yeast fermenting sugars), mixed microbial cultures in traditional foods.

  • Why: Works even without knowing genetics; relies on the organism’s native pathways.

• Molecular biotechnology: Edits/controls genes and molecular pathways inside cells.
  

  • Examples: Adding GFP to bacteria to make them glow; producing human proteins (insulin) in microbes; recombinant vaccines.

  • Why: Gives precision—pick the gene, choose the host, control expression, purify product.

Why are bacteria and yeast common biotech hosts?

• Bacteria (prokaryotes):
  

  • How: Grow fast, cheap media, simple genetics; ideal for cloning DNA and expressing many proteins.

  • Why: Short generation time → quick iterations and high yields.

• Yeast (eukaryotes):
  

  • How: Perform post-translational modifications (glycosylation, folding) that bacteria can’t; widely regarded as safe for food/medicines.

  • Why: Better for human-like proteins (e.g., vaccine antigens) that need eukaryotic processing.

What is a plasmid and why is it used as a vector?

• Plasmid: Small, circular, extra-chromosomal DNA in bacteria (also usable in yeast). Carries non-essential but helpful genes (e.g., antibiotic resistance, stress tolerance).

• Why a vector:
  

  • Self-replicates (origin of replication) → many copies per cell.

  • Has selectable markers → easy to find modified cells.

  • Contains a multiple cloning site (MCS) → convenient DNA insertion.

List the 3 steps of molecular cloning

1. Recombinant DNA preparation:
   

   • What: Cut the plasmid and the foreign gene with restriction enzymes; ligate with DNA ligase → recombinant plasmid.

   • Why: Builds a DNA construct the host can copy/express.

1. Transform & screen:
   

   • What: Introduce plasmid into host cells (transformation). Select positives by antibiotic resistance or blue/white color test; confirm with PCR.

   • Why: Only a fraction of cells take up the plasmid—screening isolates the right ones.

1. Copy & express:
   

   • What: Expand the positive clone; express the protein/DNA; purify.

   • Why: Scale yields for product (vaccine antigen, enzyme, reporter).

How is transformation screened or confirmed?

• Selectable marker (survival): Grow on antibiotic plates; only transformed cells with the resistance gene survive.

• Blue–white screening (reporter): Insert disrupts lacZα → white colonies have the insert; blue do not.

• PCR confirmation: Amplify the target insert from colony DNA to verify presence/size.

• Why: Ensures you keep only true positives before scaling expression.

Why are archaeal enzymes important for PCR?

• What: PCR cycles between high/low temperatures; ordinary polymerases denature.

• Archaeal solution: Thermostable DNA polymerases from extremophile Archaea (live in hot vents) survive 95 °C denaturation and work at elevated temps.

• Why: Enables reliable, automated DNA amplification for screening and many downstream workflows.

Describe how the Hepatitis B recombinant vaccine is made.

• Target antigen: Viral HBsAg (surface protein)—the first epitope the immune system “sees.”

• Build: Clone HBsAg gene into a yeast plasmid (vector with promoter/marker).

• Transform & select: Genetically modify yeast; select positives; confirm (PCR/assay).

• Express & purify: Grow engineered yeast to express HBsAg protein; purify antigen stringently to remove yeast components.

• Immunization: Inject purified HBsAg → body makes specific antibodies → protects against future infection.

• Why yeast: Eukaryotic processing yields a safe, properly folded antigen.

What is one major risk of genetic modification in microbes?

• Horizontal gene transfer: Engineered antibiotic resistance or other traits on plasmids can spread to environmental microbes

• GMO escape/ecology: Modified organisms might persist or alter ecosystems (competition, gene flow).

• Product purity/safety: Inadequate purification can leave host DNA/proteins in medical products.

• Why addressed: Biosafety, containment, rigorous purification and regulatory oversight are integral to responsible biotech.