Pocket Notes — Biotechnology: Principles and Processes
Definition (EFB)
Integration of natural sciences + engineering for tech applications using living organisms/cells.
Three requirements of rDNA
- Isolate desired gene.
- Insert using a vector.
- Maintain & multiply in host.
Restriction enzymes
- Restriction endonucleases cut DNA at palindromic sequences.
- First: Hind II (Smith & Nathans, 1978 Nobel).
- EcoRI — E. coli R strain (first enzyme).
- Cut styles: blunt or sticky ends (short 5' overhangs).
- DNA ligase — seals nicks / joins fragments.
Palindrome example (EcoRI)
`` 5' G A A T T C 3' 3' C T T A A G 5' `` Cut between G and A on both strands → sticky end.
PCR essentials
| Component | Role |
|---|---|
| Template DNA | The sequence to be copied |
| Primers | Short DNA that starts synthesis |
| dNTPs | Building blocks |
| Taq polymerase | Heat-stable enzyme (from Thermus aquaticus) |
| Buffer + Mg²⁺ | Environment |
Cycle steps:
- Denaturation ~94-95°C
- Annealing ~50-60°C
- Extension ~72°C
30 cycles → ~10⁹ copies.
Vector features (must-have checklist)
- Ori (origin of replication) — for copying.
- Selectable marker — antibiotic resistance genes (amp^R, tet^R).
- Cloning sites — restriction enzyme recognition sites.
- Small size — easier manipulation.
Common vector: pBR322 — plasmid with amp^R, tet^R.
Insertional inactivation
- If foreign gene inserted inside tet^R gene, host loses tet resistance.
- Grow on amp → amp^R colonies (all with plasmid).
- Replica plate on tet → tet-sensitive colonies = recombinants.
Making competent cells
- Ca²⁺ ion treatment + heat shock at 42°C briefly + ice.
- Uptake efficiency increases dramatically.
Other transformation methods
- Microinjection — direct into nucleus (animal cells).
- Gene gun / biolistics — gold particles coated with DNA (plants).
- Disarmed pathogen vectors — Agrobacterium (plants), retroviruses (animal).
rDNA workflow (memorise steps)
- Isolation of DNA
- Fragmentation by restriction enzymes
- Isolation of the target fragment
- Amplification (PCR / cloning)
- Ligation into vector
- Transformation of host
- Selection of recombinant colonies
- Product formation + downstream processing
Cell wall breakers by organism
| Organism | Enzyme |
|---|---|
| Bacteria | Lysozyme |
| Plants | Cellulase |
| Fungi | Chitinase |
DNA precipitation
Add chilled ethanol → DNA appears as collectible white threads.
Bioreactor types
- Stirred-tank reactor — most common.
- Sparged stirred-tank — sterile air blown via sparger.
- Capacity: 100 – 1000 L.
Downstream processing steps
- Separation → purification → formulation → QC tests.
MCQ traps
- Which enzyme joins fragments? — DNA ligase.
- Enzyme in PCR? — Taq polymerase (from T. aquaticus).
- Chemical for competent cells? — CaCl₂ / Ca²⁺.
- pBR322's markers? — amp^R and tet^R.
- Where is pBR322 from? — E. coli.