Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. The European Federation of Biotechnology (EFB) has given a definition encompassing both traditional views and modern molecular biotechnology: “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”

Principles of Biotechnology:

1. Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism.

2. Bioprocess Engineering: Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities.

   The construction of the first recombinant DNA (rDNA) emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium. This was accomplished by Stanley Cohen and Herbert Boyer in 1972.

Tools of Recombinant DNA Technology:

• Restriction Enzymes (Molecular Scissors): Hind II was the first restriction endonuclease to be isolated. It always cut DNA at a specific point by recognizing a specific sequence of six base pairs (recognition sequence). Today, we know more than 900 restriction enzymes.

  • Naming: First letter from Genus, second two from species (e.g., EcoRI comes from Escherichia coli RY 13). ‘R’ is the strain, ‘I’ is the order of isolation.

  • Mechanism: Restriction endonucleases inspect the DNA length and bind to specific recognition sequences. They cut the sugar-phosphate backbones. EcoRI recognizes the Palindrome sequence 5′-GAATTC-3′. A palindrome reads the same on the two strands when orientation is kept the same.

  • Sticky Ends: Enzymes cut slightly away from the center of the palindrome, leaving single-stranded portions at the ends called “sticky ends”. These facilitate the action of the enzyme DNA ligase.

• Cloning Vectors: Plasmids and bacteriophages are used. Features required to facilitate cloning:

  1. Origin of Replication (ori): Sequence where replication starts. Also controls the copy number of the linked DNA.

  2. Selectable Marker: Helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants. Usually, genes encoding resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, or kanamycin are useful markers for E. coli.

  3. Cloning Sites: Recognition sites for restriction enzymes. To avoid complications, the vector should have very few (preferably single) recognition sites. pBR322 is a widely used E. coli cloning vector. It has restriction sites (e.g., BamH I, Sal I in tetracycline resistance gene).

     • Insertional Inactivation: If a foreign DNA is ligated at the BamH I site of the tetracycline resistance gene, the recombinant plasmid loses tetracycline resistance. In Blue-White screening, recombinant DNA is inserted within the coding sequence of an enzyme, $\beta$-galactosidase. This results in inactivation of the enzyme. Colonies with non-recombinant plasmids produce a blue colour (due to chromogenic substrate), while recombinants produce white colonies.

  4. Vectors for Plants and Animals: Agrobacterium tumefaciens delivers a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor. The Ti plasmid is now modified into a cloning vector which is no longer pathogenic. Retroviruses in animals transform normal cells into cancerous cells; they are now used as vectors to deliver desirable genes.

• Competent Host: Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. Bacterial cells are treated with a specific concentration of a divalent cation like Calcium, which increases the efficiency of DNA entry. Recombinant DNA can then be forced into cells by incubating on ice, followed by a brief Heat Shock (42°C), and back on ice.

  • Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.

  • Biolistics or Gene Gun: Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA (suitable for plants).

Processes of Recombinant DNA Technology:

1. 1. Isolation of Genetic Material: Cells are treated with enzymes like Lysozyme (bacteria), Cellulase (plant cells), or Chitinase (fungus) to break the cell wall. RNA is removed by Ribonuclease, proteins by Protease. Purified DNA precipitates out after adding chilled ethanol and can be removed by spooling.

   2. Cutting of DNA: Performed by restriction enzymes.

   3. Separation of DNA Fragments: Gel Electrophoresis. DNA fragments are negatively charged and move towards the anode through a matrix (usually agarose, a natural polymer extracted from sea weeds). Smaller fragments move farther. The separated DNA fragments can be visualized only after staining with Ethidium bromide followed by exposure to UV radiation (appear as bright orange bands). The extraction of DNA from the gel piece is called Elution.

   4. Amplification of Gene of Interest (PCR): Polymerase Chain Reaction. Multiple copies of the DNA are synthesized in vitro using two sets of primers and the enzyme DNA polymerase.

      • Steps: Denaturation (heating), Annealing (primers bind), and Extension (polymerase adds nucleotides).

      • Thermostable DNA polymerase (isolated from bacterium Thermus aquaticus) is used, which remains active at high temperatures (induced denaturation).

5. Ligation: Joining the gene of interest to the vector.

6. Transformation: Introduction of recombinant DNA into the host.

7. Obtaining the Foreign Gene Product: The ultimate aim is to produce a desirable protein. Cultures can be grown in large volumes (100-1000 litres) in Bioreactors.

   • Stirred-tank bioreactor: Usually cylindrical or with a curved base to facilitate mixing.

   • Sparged stirred-tank bioreactor: Sterile air is sparged (bubbled) through the reactor. Bioreactors have an agitator system, oxygen delivery system, foam control system, temperature control system, pH control system, and sampling ports.

8. Downstream Processing: Includes separation and purification of the biosynthetic product. The product is formulated with suitable preservatives and undergoes strict quality control testing before marketing.