Key Points

Biotechnology involves using live organisms, cells, or their components to create products and processes useful to humans. The European Federation of Biotechnology (EFB) defines it as 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Modern biotechnology is based on two core techniques: Genetic engineering, which alters the chemistry of genetic material (DNA/RNA), and Bioprocess engineering, which maintains a sterile environment for large-scale culture of desired cells.

Genetic engineering involves creating recombinant DNA (rDNA) by isolating and introducing desirable genes into a host organism. This overcomes the limitations of traditional hybridization by avoiding the transfer of undesirable genes.

In 1972, Stanley Cohen and Herbert Boyer constructed the first artificial recombinant DNA molecule. They linked an antibiotic resistance gene to a native plasmid of Salmonella typhimurium and transferred it into E. coli.

Restriction enzymes, or restriction endonucleases, cut DNA at specific recognition sites. These enzymes are crucial for creating DNA fragments for recombination. For example, EcoRI is isolated from Escherichia coli RY 13.

Restriction enzymes recognize and cut specific palindromic nucleotide sequences, which read the same on both strands in the $5' \\rightarrow 3'$ direction. The cut often produces overhanging 'sticky ends' that facilitate joining DNA fragments via DNA ligase.

Cloning vectors, like plasmids and bacteriophages, are DNA molecules that carry foreign DNA into a host cell and can replicate independently. They act as vehicles to multiply the gene of interest within the host.

A cloning vector must have an origin of replication (ori) to control copy number, a selectable marker to identify transformants, and one or more unique cloning sites (restriction sites) for inserting foreign DNA.

Selectable markers, like antibiotic resistance genes (e.g., $amp^R$, $tet^R$ in plasmid pBR322), help in selecting transformed cells. Non-transformed cells cannot grow in the presence of the corresponding antibiotic.

This method differentiates recombinant from non-recombinant colonies. Inserting foreign DNA into the coding sequence of an enzyme like $\\beta$-galactosidase inactivates it, preventing color production on a chromogenic substrate and identifying recombinant colonies.

PCR is a technique to amplify a specific DNA segment in vitro, creating billions of copies. It requires two sets of primers, nucleotides, and a thermostable DNA polymerase, such as Taq polymerase from Thermus aquaticus.

A single PCR cycle consists of three steps: Denaturation (heating to separate DNA strands, typically at $94-96^{\\circ}\\text{C}$), Annealing (cooling to allow primers to bind to DNA), and Extension (DNA polymerase synthesizes new strands).

Bacterial cells are made 'competent' to take up foreign DNA by treating them with a divalent cation like calcium ($Ca^{2+}$). A subsequent heat shock at $42^{\\circ}\\text{C}$ forces the cells to take up the recombinant DNA.

Besides making hosts competent, other methods include micro-injection (direct injection into an animal cell nucleus), biolistics or gene gun (bombarding plant cells with DNA-coated micro-particles), and using disarmed pathogen vectors.

To isolate pure DNA, cells are treated with enzymes like lysozyme (bacteria), cellulase (plants), or chitinase (fungi). RNA and proteins are removed using ribonuclease and protease, respectively, and purified DNA is precipitated with chilled ethanol.

This technique separates DNA fragments based on their size. Since DNA is negatively charged, it moves towards the positive electrode (anode) through an agarose gel matrix. Smaller fragments move farther than larger ones.

Bioreactors are large vessels (100-1000 litres) used to culture cells for large-scale production of recombinant proteins. They provide optimal growth conditions like temperature, pH, oxygen, and nutrients.

After the biosynthetic stage in a bioreactor, the product undergoes downstream processing. This includes separation, purification, formulation with preservatives, and strict quality control testing before it is ready for marketing.