Biology: Class 12 — Chapter 9: Biotechnology : Principles and Processes

• 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.
• 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 for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

Asexual reproduction preserves genetic information, while sexual reproduction permits variation. Traditional hybridisation often leads to the inclusion and multiplication of undesirable genes along with desirable ones. Genetic engineering overcomes this limitation by allowing the isolation and introduction of only desirable genes.

Key concepts in gene cloning include:

• Origin of replication (ori): A specific DNA sequence in a chromosome responsible for initiating replication. An alien DNA must be linked to this sequence to replicate inside the host.
• Cloning: The process of making multiple identical copies of any template DNA.
• First Recombinant DNA: Constructed by Stanley Cohen and Herbert Boyer in 1972. They isolated an antibiotic resistance gene from a plasmid of Salmonella typhimurium and linked it to a plasmid vector, which was then transferred into Escherichia coli.

Three basic steps in genetically modifying an organism:

1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host.
3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Discovered in 1963 in E. coli as enzymes restricting bacteriophage growth. One added methyl groups to DNA, while the other cut DNA (called restriction endonuclease).

• The first restriction endonuclease isolated was Hind II, which cuts DNA at a specific six-base-pair sequence called the recognition sequence.
• Today, more than 900 restriction enzymes have been isolated from over 230 strains of bacteria.
• Naming Convention: The first letter comes from the genus, the next two from the species of the prokaryotic cell (e.g., EcoRI from Escherichia coli RY 13), and the Roman number indicates the order of isolation.
• Classification: Restriction enzymes belong to a larger class called nucleases:
  • Exonucleases: Remove nucleotides from the ends of the DNA.
  • Endonucleases: Make cuts at specific positions within the DNA.
• Palindromic Nucleotide Sequences: Restriction endonucleases recognize specific palindromic sequences where the sequence of base pairs reads the same on both strands when the orientation of reading is kept the same (5′ → 3′ or 3′ → 5′). For example:

          5′ —— GAATTC —— 3′
          3′ —— CTTAAG —— 5′
          

• Sticky Ends: Enzymes cut the strands a little away from the center of the palindrome sites, leaving single-stranded overhanging stretches. These hydrogen-bond with their complementary counterparts, facilitating the action of DNA ligase.

Gel Electrophoresis: A technique used to separate DNA fragments based on size.

• Since DNA fragments are negatively charged, they move towards the positive electrode (anode) under an electric field through a matrix of agarose (a natural polymer from sea weeds).
• The smaller the fragment size, the farther it moves through the sieving effect of the gel.
• Visualization: DNA fragments can only be seen after staining with ethidium bromide followed by exposure to UV radiation, appearing as bright orange bands.
• Elution: The process of cutting out and extracting the separated bands of DNA from the agarose gel.

Plasmids and bacteriophages are commonly used as vectors because of their ability to replicate independently of chromosomal DNA control within bacterial cells.

Essential Features of a Cloning Vector:

• Origin of replication (ori): Controls replication and determines the copy number of the linked DNA.
• Selectable marker: Helps identify and eliminate non-transformants and selectively permit the growth of transformants. Examples for E. coli include genes conferring resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, or kanamycin.
• Cloning sites: Recognition sites for restriction enzymes where foreign DNA is ligated. Single or very few recognition sites are preferred. For example, in vector pBR322, foreign DNA can be ligated at the BamH I site of the tetracycline resistance gene.
• Insertional Inactivation: A selection method where a recombinant DNA is inserted within the coding sequence of an enzyme (like β-galactosidase), inactivating the gene. Recombinants do not produce color in the presence of a chromogenic substrate, whereas non-recombinants form blue colonies.
• Vectors for Plants and Animals:
  • Plants: The tumor-inducing (Ti plasmid) of Agrobacterium tumefaciens has been modified into a non-pathogenic cloning vector to deliver genes of interest.
  • Animals: Retroviruses have been disarmed and are used to deliver desirable genes into animal cells.

Since DNA is hydrophilic, it cannot pass through cell membranes. Cells must be made "competent" to take up DNA using various methods:

• Chemical Treatment: Treating bacterial cells with a specific concentration of a divalent cation (such as calcium) followed by heat shock (incubating on ice, then briefly at 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).
• Disarmed Pathogen Vectors: Allowed to infect the cell and transfer the recombinant DNA.

To obtain pure DNA free from other macromolecules, cells are treated with specific enzymes:

• Lysozyme: For bacteria.
• Cellulase: For plant cells.
• Chitinase: For fungi.
• RNA and Protein Removal: Treated with ribonuclease and protease respectively.
• Precipitation: Purified DNA precipitates out as fine threads after adding chilled ethanol and is removed by spooling.

DNA is cut at specific locations using restriction enzymes, and the gene of interest is amplified using Polymerase Chain Reaction (PCR).

Three Steps of PCR:

1. Denaturation: High temperature separates the double-stranded DNA.
2. Annealing: Two sets of primers bind to the single-stranded DNA templates.
3. Extension: Thermostable Taq polymerase (isolated from the bacterium Thermus aquaticus) extends the primers using deoxynucleotides.

Repeated cycles can amplify the DNA segment approximately 1 billion times.

• Insertion: Recombinant DNA is introduced into the host cell (e.g., using ampicillin resistance as a selectable marker).
• Recombinant Protein: Any protein-encoding gene expressed in a heterologous host.
• Bioreactors: Large vessels (100–1000 liters) used for large-scale production of biological products under optimal conditions (temperature, pH, substrate, salts, vitamins, oxygen).
• Types of Bioreactors:
  • Simple stirred-tank bioreactor: Facilitates even mixing and oxygen availability.
  • Sparged stirred-tank bioreactor: Sterile air bubbles are sparged to increase the surface area for oxygen transfer.

The processes that occur after the biosynthetic stage to prepare the product for marketing:

• Separation and purification of the product.
• Formulation with suitable preservatives.
• Clinical trials (especially for drugs) and strict quality control testing.