🧬 Biotechnology: Principles and Processes Study Notes

9.1 1. What is Biotechnology?

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

The EFB (European Federation of Biotechnology) definition covers both traditional and modern forms: "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services."

Traditional vs Modern

9.2 2. Principles of Biotechnology

The two founding principles are genetic engineering and maintenance of sterile conditions in chemical engineering processes (bioprocessing).

The first recombinant DNA

Stanley Cohen and Herbert Boyer (1972) made the first artificial recombinant DNA by isolating an antibiotic resistance gene (cut from a plasmid using a restriction enzyme) and linking it with a native plasmid of Salmonella typhimurium. The plasmid acts as a vector that carries and multiplies the linked (alien) DNA.

Three basic steps in genetically modifying an organism

9.3 3. Tools I — Restriction Enzymes

Key tools of rDNA technology: restriction enzymes, polymerases, ligases, vectors, and a host organism.

Discovery

• In 1963, two enzymes restricting bacteriophage growth in E. coli were found: one added methyl groups (methylase), the other cut DNA — the restriction endonuclease.
• Hind II = first restriction endonuclease; always cuts DNA at a specific point by recognising a specific sequence of 6 base pairs (the recognition sequence).
• Today >900 restriction enzymes are known from over 230 bacterial strains.

Nomenclature — example EcoRI

Nucleases

Palindrome & sticky ends

A palindrome reads the same on both strands in the same (5'→3') orientation, e.g. EcoRI site:

The enzyme cuts a little away from the centre, between the same two bases on both strands, leaving overhanging single-stranded sticky ends. Fragments cut by the same enzyme have complementary sticky ends and are joined by DNA ligase.

9.4 4. Tools II — Cloning Vectors

Plasmids and bacteriophages replicate within bacteria independent of chromosomal DNA and are used as vectors. Alien DNA linked to them is multiplied to the copy number of the vector.

Features a good vector needs

Insertional inactivation

pBR322 has two resistance genes: ampR and tetR. Inserting alien DNA into the BamHI site of tetR inactivates tetracycline resistance. Recombinants grow on ampicillin but are tet-sensitive — but this needs two plates (cumbersome).

Blue–white selection (better)

Insert DNA into the coding sequence of enzyme β-galactosidase → inactivates it.

Vectors for plants & animals

• Ti plasmid of Agrobacterium tumefaciens (a dicot pathogen) delivers T-DNA; disarmed and used to transform plant cells.
• Retroviruses in animals (normally transform cells to cancerous) are disarmed to deliver genes into animal cells.

9.5 5. Tools III — Making a Competent Host

DNA is hydrophilic and cannot cross the cell membrane. So the host must be made competent to take up DNA.

Chemical (heat-shock) method

Ca²⁺ increases pore efficiency in the cell wall so DNA enters.

Other delivery methods

9.6 6. Separation & Isolation of DNA Fragments

Agarose gel electrophoresis separates DNA fragments by size.

• DNA is negatively charged → moves toward the anode (+) through the agarose matrix.
• Smaller fragments move farther; larger ones lag near the wells.
• Stained with ethidium bromide, visualised as bright orange bands under UV light (invisible in visible light).

9.7 7. Processes of rDNA Technology

The full workflow has the following ordered steps:

(a) Isolation of genetic material

Break cells open using enzymes: lysozyme (bacteria), cellulase (plant), chitinase (fungi). Remove RNA with ribonuclease, proteins with protease. Add chilled ethanol → DNA precipitates as fine collectable threads.

(b) Cutting DNA

Incubate purified DNA with restriction enzyme at optimal conditions; check progress by gel electrophoresis; cut the vector with the same enzyme; mix + ligase → recombinant DNA.

(c) Amplification by PCR

Make many copies in vitro using two primers and DNA polymerase.

Repeated cycles amplify DNA to ~1 billion (10⁹) copies. Uses Taq polymerase from Thermus aquaticus — thermostable, survives the high-temp denaturation.

(d) Insertion into host

rDNA (e.g. carrying an ampicillin-resistance marker) is put into competent E. coli; only transformants grow on ampicillin plates → marker enables selection.

(e) Obtaining the foreign gene product

A protein made in a heterologous host = recombinant protein. Grown in a continuous culture system (fresh medium in, spent medium out) to keep cells in the active log phase for higher yield; scaled up in a bioreactor.

9.8 8. Bioreactors & Downstream Processing

Bioreactors are vessels where raw materials are biologically converted into products/enzymes using microbial, plant, animal or human cells. They provide optimal temperature, pH, substrate, salts, vitamins and oxygen.

Types of stirred-tank bioreactor

A bioreactor has

• Agitator system
• Oxygen delivery system
• Foam control system
• Temperature & pH control systems
• Sampling ports (withdraw small culture volumes periodically)