Biotechnology – Advanced Study Notes
B.Pharm Admission – Biology
(MIT‑level, first‑principles approach)
1. Introduction
- Biotechnology: Application of living organisms or their molecular components to develop products or processes that benefit humanity.
- Scope: From drug discovery to bio‑fuel, from gene therapy to synthetic biology.
- Historical Milestones:
- 1973 – Recombinant DNA (Sanger, Novick).
- 1983 – First recombinant protein drug (Humulin).
- 2003 – Human genome project completion.
- 2012 – CRISPR‑Cas9 gene editing demonstration.
2. Molecular Foundations
| Process | Key Players | Core Mechanisms |
|---|---|---|
| DNA replication | DNA polymerase, helicase, primase | Semi‑conservative replication; Okazaki fragments |
| Transcription | RNA polymerase, promoter, enhancer, transcription factors | Initiation, elongation, termination |
| Translation | Ribosome, tRNA, mRNA, release factors | Codon‑anticodon pairing, peptide bond formation |
| Post‑translational modification | Kinases, phosphatases, glycosyltransferases | Regulation of activity, stability, localization |
First principles: These processes are governed by thermodynamics (free‑energy landscapes) and kinetics (rate constants). For instance, the Gibbs free energy change for nucleotide addition is ~−1.5 kcal/mol, ensuring directional synthesis.
3. Recombinant DNA Technology
3.1 Vector Design
- Plasmids: Circular, supercoiled DNA; size 1–10 kb.
- Key Elements
- Origin of replication (ori) – dictates copy number.
- Selectable marker – antibiotic resistance, auxotrophy.
- Promoter – constitutive (lac, T7) vs. inducible (lacI, TetO).
- Multiple cloning site (MCS) – unique restriction enzymes.
- Terminator – transcription termination signals.
3.2 Host Cells
| Host | Advantages | Limitations |
|---|---|---|
| E. coli | Fast growth, high yield, inexpensive | Lack post‑translational modifications, endotoxin |
| Yeast (S. cerevisiae) | Eukaryotic PTM, scalable | Lower protein yield, glycosylation patterns |
| Mammalian cells (CHO, HEK293) | Human‑like PTM, regulated secretion | Slow growth, high cost |
| Bacillus subtilis | Secretion into medium, no endotoxin | Limited PTM |
Mathematical Model:
[ \frac{dP}{dt} = \frac{k_{\text{trans}} \cdot \text{mRNA}}{1 + \frac{P}{K_{\text{deg}}}} - k_{\text{deg}}\cdot P ] where (P) = protein concentration, (k_{\text{trans}}) transcription/translation rate, (k_{\text{deg}}) degradation constant.
3.3 Gene Assembly Strategies
- Restriction Enzyme Cloning – classical, but limited by restriction sites.
- Gibson Assembly – overlapping fragments, no restriction.
- Golden Gate – Type IIS enzymes, modular assembly.
- CRISPR‑mediated knock‑ins – HDR or NHEJ dependent.
4. Gene Editing: CRISPR‑Cas Systems
4.1 Core Components
| Component | Function |
|---|---|
| Cas9 | DNA endonuclease, guided by sgRNA |
| sgRNA | 20‑nt spacer + scaffold |
| PAM | 5’‑NGG‑3’ (for SpCas9) required for cleavage |
4.2 Mechanism
- sgRNA binds Cas9 → RNP complex.
- RNP scans DNA for PAM → R-loop formation.
- Dual‑strand break (DSB) 3 bp upstream of PAM.
- Repair:
- NHEJ → indels (knock‑out).
- HDR → precise edits (knock‑in).
4.3 Advanced Variants
| Variant | Feature | Use‑case |
|---|---|---|
| dCas9 | Catalytically dead | Gene repression (CRISPRi) or activation (CRISPRa) |
| Base Editors | A→G (adenine) or C→T (cytosine) | Precise point mutations without DSB |
| Prime Editors | Reverse transcriptase + pegRNA | Insertions, deletions, all 12 base transitions |
| CRISPR‑Cas12a (Cpf1) | PAM TTTV, staggered cuts | Higher multiplexing, less off‑target |
Edge Cases
- Mosaicism: In vivo editing may produce heterogeneous cell populations.
- Off‑target cleavage: Requires careful sgRNA design (e.g., using CRISPOR).
- Immunogenicity: Cas proteins can trigger innate immunity in humans.
5. Gene Therapy & Delivery Vectors
| Vector | Pros | Cons | Typical Use |
|---|---|---|---|
| Adeno‑Associated Virus (AAV) | Low immunogenicity, long‑term expression | Limited cargo (∼4.7 kb) |