BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Modern biotechnology combines genetic engineering with bioprocess engineering to make useful products at scale from modified organisms, cells or enzymes.

Modern biotechnology rests on two core techniques: genetic engineering, which alters and transfers DNA/RNA, and bioprocess engineering, which maintains sterile conditions for producing desired cells or products in quantity.

• Genetic engineering avoids the unwanted genes often carried along in traditional hybridisation. It enables transfer of one or a chosen set of desirable genes.
• The three basic steps are identifying DNA with desirable genes, introducing it into a host, and ensuring its maintenance and inheritance in progeny.
• A recombinant DNA molecule is made in vitro by joining DNA from different sources. It can replicate when linked to a suitable vector or integrated into host genome.

The essential tools are restriction enzymes, DNA polymerases, ligases, cloning vectors and a suitable host organism.

• Restriction endonucleases recognise specific palindromic DNA sequences and cut both strands. EcoRI recognises 5'-GAATTC-3' and leaves sticky ends.
• When source DNA and vector are cut with the same restriction enzyme, their complementary sticky ends can pair. DNA ligase seals the backbone to form recombinant DNA.
• DNA fragments are separated by agarose gel electrophoresis. DNA moves towards the anode; smaller fragments travel farther. Ethidium bromide staining and UV reveal bands, and elution recovers a desired band.

A useful vector has an origin of replication (ori), selectable markers and preferably a single cloning site for a restriction enzyme.

• ori starts replication and controls copy number. Plasmids and bacteriophages replicate independently of bacterial chromosomal DNA.
• Antibiotic-resistance genes can select transformants. In pBR322, insertion at the BamHI site in the tetracycline-resistance gene inactivates that marker.
• Blue-white screening uses insertional inactivation of beta-galactosidase. Non-recombinant colonies are blue with chromogenic substrate; recombinant colonies are colourless.
• Agrobacterium tumefaciens and retroviruses inspired vector systems for gene delivery into plant and animal cells, respectively.

Host cells must be made competent to receive DNA. Bacterial cells can be treated with divalent cations, incubated with DNA on ice, exposed briefly to 42 C heat shock and returned to ice.

• Microinjection directly injects recombinant DNA into an animal-cell nucleus.
• Biolistics, or gene gun, delivers DNA-coated gold or tungsten particles into plant cells.
• Disarmed pathogen vectors can transfer recombinant DNA into host cells without causing disease.

The workflow is isolation of DNA, restriction digestion, isolation of desired fragment, ligation into vector, transfer into host, large-scale culture and extraction of product.

• DNA must be purified from proteins, RNA and other macromolecules before restriction digestion.
• After the desired gene and vector are cut with the same enzyme, ligase joins them to prepare recombinant DNA.
• Transformation is confirmed by selectable markers: cells gaining an ampicillin-resistance gene grow on ampicillin while non-transformants do not.

Polymerase chain reaction synthesises many copies of a selected DNA segment in vitro using two primers, nucleotides, template DNA and DNA polymerase.

• Each cycle has three stages: denaturation of double-stranded DNA, primer annealing and primer extension.
• Taq polymerase from Thermus aquaticus remains active after high-temperature denaturation.
• Repeated cycles can make approximately one billion copies of the target segment, which can then be cloned if needed.

Expression of the foreign gene produces the desired protein or other product. Small cultures yield little material, so commercial production uses bioreactors of about 100-1000 litres.

• Stirred-tank bioreactors are cylindrical or have a curved base. Their agitator ensures mixing and oxygen availability; sterile air may be sparged through the culture.
• They provide control of temperature, pH, substrate, salts, vitamins, oxygen, foam and periodic sampling.
• Downstream processing includes separation, purification, formulation and strict quality control before marketing, especially for therapeutic products.

• Restriction endonucleases create defined DNA fragments; ligase joins compatible ends.
• Vectors need ori, selectable markers and suitable cloning sites.
• Gel electrophoresis separates DNA by size; PCR amplifies a chosen DNA sequence.
• Competent host cells take up recombinant DNA and are selected using markers.
• Bioreactors enable large-scale production; downstream processing prepares the product for use.