Biotechnology: Principles and Processes — Long Notes

Biotechnology applies living organisms/cells or their components to make useful products and processes. The European Federation of Biotechnology (EFB) defines it as the integration of natural sciences and engineering for the application of organisms, cells, or their parts and molecular analogues to products and services.

Modern biotechnology has three core pillars:

1. Recombinant DNA technology (genetic engineering).
2. Chemical engineering — bioreactors, sterile conditions, downstream processing.
3. Cell culture and tissue culture.

1. Principles of Genetic Engineering

Genetic engineering allows manipulation of DNA in ways that would be impossible for natural mating. Three basic principles enable it:

1. Identification of DNA with the desirable gene.
2. Introduction into a suitable host with a vector.
3. Maintenance and multiplication of introduced DNA in host; selection of transformants carrying the recombinant DNA.

2. Tools of Recombinant DNA Technology

2.1 Restriction Enzymes (Molecular Scissors)

• Restriction endonucleases cut DNA at specific recognition sequences, typically 4-8 bp palindromic sequences.
• Discovered by W. Arber, H. Smith, D. Nathans — Nobel Prize 1978.
• The first enzyme, Hind II, was isolated from Haemophilus influenzae Rd strain.
• Naming: EcoRI = *Escherichia coli R strain, first (I) enzyme discovered.

Palindrome — reads the same in both directions (5'→3' on both strands). Example (EcoRI): `` 5' G A A T T C 3' 3' C T T A A G 5' `` EcoRI cuts between G and A on each strand → creates sticky ends — short single-stranded overhangs that can base-pair with complementary overhangs from another cut piece.

Some enzymes leave blunt ends (no overhang) — harder to ligate.

DNA ligase — seals the phosphodiester backbone between two DNA pieces held together by sticky ends.

Both restriction enzymes and ligase are essential for making recombinant DNA.

2.2 Vectors

Carriers that transport foreign DNA into a host cell. Common types:

• Plasmids — small (few kb), circular, extra-chromosomal DNA in bacteria; replicate independently.
• Bacteriophages — viruses that infect bacteria; carry cloned DNA into bacterial cells.

A useful vector must have:

• Origin of replication (ori) — allows autonomous copying.
• Selectable marker — a gene (usually antibiotic resistance) to identify transformed cells.
• Cloning sites — a small region with unique restriction sites where the foreign DNA can be inserted.
• Small size — for easy manipulation and high copy number.

pBR322 is a widely used cloning vector — an E. coli plasmid with amp^R and tet^R antibiotic resistance genes and multiple restriction sites for cloning.

Insertional Inactivation

If the foreign gene is inserted within an antibiotic resistance gene (say, tet^R), that gene is inactivated. Cells with a plasmid carrying the insert will:

• Still grow on amp (amp^R intact).
• Fail to grow on tet (tet^R disrupted).

This lets us pick out true recombinant colonies (those carrying the foreign DNA) from just-plasmid colonies.

Modern vectors use chromogenic insertional inactivation (blue-white screening) as an even easier readout.

2.3 Competent Host

Bacteria don't naturally take up foreign DNA. We must make them competent to allow this:

• Treatment with divalent Ca²⁺ ions followed by a brief heat shock at ~42°C and rapid cooling on ice.
• This creates transient pores in the bacterial membrane allowing DNA entry.

Other methods for other organisms:

• Microinjection — needle injection into animal cell nucleus.
• Gene gun (biolistics) — high-velocity gold or tungsten microparticles coated with DNA are shot into plant cells.
• Disarmed pathogen vectors — e.g. Agrobacterium tumefaciens for plants; modified retroviruses for animal cells.

2.4 Polymerase Chain Reaction (PCR)

Amplifies a specific DNA fragment a billion-fold in a few hours. Requires:

• Template DNA.
• Two primers flanking the target region.
• dNTPs as building blocks.
• Taq DNA polymerase — a thermostable enzyme from Thermus aquaticus — survives 90°C+ temperatures.
• Buffer with Mg²⁺.

Cycle steps (repeated 25–35 times):

1. Denaturation at ~94-95°C — double-stranded DNA separates.
2. Annealing at ~50-60°C — primers bind their complementary sequences.
3. Extension at ~72°C — Taq polymerase extends from primers.

Each cycle doubles the target DNA. After 30 cycles → ~10⁹ copies. PCR revolutionised molecular biology, medicine, forensics, and evolutionary studies.

3. Processes of Recombinant DNA Technology

Step 1: Isolation of Genetic Material (DNA)

• Bacteria — cell walls broken by lysozyme.
• Plants — walls broken by cellulase.
• Fungi — walls broken by chitinase.
• Proteins are removed by protease; RNA by ribonuclease.
• DNA precipitates as a fine white thread on adding chilled ethanol — can be collected by spooling on a glass rod.

Step 2: Cutting DNA at Specific Locations

Restriction enzymes digest DNA into fragments; agarose gel electrophoresis separates fragments by size. Smaller fragments move faster. Visualisation with ethidium bromide under UV.

The desired fragment is cut out (eluted) from the gel for use.

Step 3: Amplification of DNA using PCR

Alternatively (or in addition), the fragment is amplified by PCR before cloning.

Step 4: Insertion of Recombinant DNA into the Host

• Vector cut with same enzyme as target DNA → complementary sticky ends.
• Target + vector + DNA ligase → recombinant DNA molecule.
• Recombinant DNA introduced into competent host cell by transformation (CaCl₂ method, gene gun, etc.).
• Grow on selective medium (e.g. antibiotic-containing) so only successfully transformed cells survive.
• Screen for recombinants via insertional inactivation or blue-white screening.

Step 5: Obtaining the Foreign Gene Product

• Grow transformed bacteria in nutrient medium so the recombinant gene expresses the protein.
• Small-scale in shake flasks; industrial-scale in bioreactors.

Step 6: Downstream Processing

Isolate the product from the bioreactor:

• Separation of cells and medium.
• Purification via centrifugation, filtration, chromatography, precipitation.
• Formulation with preservatives, stabilisers.
• Quality control and clinical trial checks (for therapeutic proteins).

4. Bioreactors

Large vessels (typically 100 – 1000 L) used for controlled cell/microbe culture:

• Temperature, pH, dissolved O₂, substrate levels, agitation all tuned for optimum yield.
• Sterile conditions maintained.

Types:

• Simple stirred-tank bioreactor — mechanical stirrer keeps cells and medium mixed.
• Sparged stirred-tank bioreactor — sterile air is bubbled in from the bottom (sparged) while the stirrer helps distribute it — provides good oxygen transfer.
• Fitted with agitator, foam breaker, temperature control, pH control, aeration and sampling ports.

5. Downstream Processing (Overview)

• Centrifugation / filtration — remove cells.
• Precipitation or chromatography — purify the desired molecule.
• Formulation — add preservatives, correct pH, package.
• Quality control — HPLC, bio-assays, contamination screening.

Downstream processing often costs more than upstream fermentation and is critical for drug approval.

Key take-aways

1. Modern biotech is powered by restriction enzymes + ligase + vectors + competent hosts + PCR + bioreactors.
2. Restriction enzymes cut DNA at palindromic sites and produce sticky/blunt ends that ligase can join.
3. PCR amplifies specific DNA regions using heat-stable Taq polymerase across denature-anneal-extend cycles.
4. Plasmid vectors like pBR322 must have origin, selectable markers, cloning sites, and be small.
5. Transforming bacteria uses CaCl₂ + heat shock; plants use gene gun/Agrobacterium; animal cells use microinjection/retroviruses.
6. Scale-up to bioreactors with careful downstream processing turns lab-scale rDNA into industrial and clinical products.