Chapter 9 – Biotechnology

Core Principles of Biotechnology

• Definition (EFB): The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.
• Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA) to introduce these into host organisms and change the phenotype of the host.
• Bioprocess Engineering: Maintenance of sterile (microbial contamination-free) conditions to enable the growth of only the desired microbe/eukaryotic cell in large quantities.
• The First Recombinant DNA (rDNA): * Constructed by Stanley Cohen and Herbert Boyer in 1972.
  • They isolated an antibiotic resistance gene from a plasmid of Salmonella typhi.
  • This was linked with a plasmid vector of Escherichia coli using DNA ligase.
• Basic Steps in Genetically Modifying an Organism:
  • Identification of DNA with desirable genes.
  • Introduction of the identified DNA into the host.
  • Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Tools of Recombinant DNA Technology: Enzymes

• Restriction Enzymes (Molecular Scissors):
  • Found in 1963; two enzymes responsible for restricting the growth of bacteriophage in E. coli. One added methyl groups to DNA, the other (Restriction Endonuclease) cut DNA.
  • Hind II: The first restriction endonuclease discovered. It always cuts DNA molecules at a particular point by recognizing a specific sequence of six base pairs (Recognition Sequence).
• Naming Convention: * First letter: Genus (Escherichia).
  • Second/Third letters: Species (coli).
  • Fourth letter: Strain (RY 13).
  • Roman Number: Order in which the enzymes were isolated from that strain.
• Mechanism of Action:
  • Palindromic Nucleotide Sequence: The enzyme recognizes a sequence that reads the same on the two strands when the orientation of reading (e.g., 5′ → 3′) is kept the same.
  • Sticky Ends: Enzymes cut the strand a little away from the center of the palindrome sites, leaving single-stranded overhanging stretches. These facilitate base pairing via hydrogen bonds.
• DNA Ligase: Joins the sugar-phosphate backbones of the DNA fragments to create a continuous circular rDNA.

Cloning Vectors (The Vehicles)

• Plasmids and Bacteriophages: Used as vectors because they replicate within bacterial cells independent of chromosomal DNA control.
• Essential Features for a Cloning Vector:
  1. Origin of Replication (ori): Sequence where replication starts. It is responsible for controlling the copy number of the linked DNA.
  2. Selectable Marker: Used to identify and eliminate non-transformants. Commonly used markers include genes for resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, or kanamycin.
  3. Cloning Sites: The vector needs recognition sites for restriction enzymes. If a vector has many sites for one enzyme, it will generate several fragments, complicating gene cloning.
• Selection of Recombinants (Blue-White Screening):
  • Instead of antibiotic resistance, an alternative marker is the lacZ gene (coding for beta-galactosidase).
  • If a foreign DNA is inserted within the gene, the gene is inactivated (Insertional Inactivation).
  • Non-recombinants turn blue on a chromogenic substrate; recombinants remain white.

Competent Host: For Transformation with rDNA

• Hydrophilic DNA: DNA cannot pass through cell membranes because it is water-loving (hydrophilic), and membranes are lipid-based.
• Chemical Treatment: Cells are treated with a specific concentration of a divalent cation, such as Calcium (Ca2+), which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
• Heat Shock: Incubation on ice → brief placement at 42°C → back on ice.
• Alternative Methods:
  • Micro-injection: rDNA is directly injected into the nucleus of an animal cell.
  • Biolistics (Gene Gun): High-velocity micro-particles of gold or tungsten coated with DNA are bombarded into plant cells.
  • Disarmed Pathogen Vectors: Using “disarmed” versions of Agrobacterium tumefaciens or retroviruses to transfer DNA without causing disease.

Processes of Recombinant DNA Technology

• Isolation of Genetic Material
  • Treatment with enzymes: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi).
  • RNA is removed by Ribonuclease; proteins are removed by Protease.
  • Purified DNA is precipitated by adding chilled ethanol, appearing as fine threads.
• Amplification of Gene of Interest using PCR
  • Polymerase Chain Reaction (PCR): Used to make billions of copies of a DNA segment in vitro.
  • Components: Primers (small oligonucleotides) and Taq Polymerase (thermostable enzyme from Thermus aquaticus).
• Stages:
  • Denaturation: Heating to separate the DNA strands.
  • Annealing: Primers bind to the complementary regions.
  • Extension: Polymerase adds nucleotides to the primers.

Insertion and Production

• Transformation: Recombinant DNA is introduced into a host (e.g., E. coli).
• Recombinant Protein: If a protein-encoding gene is expressed in a heterologous host.
• Bioreactors: Large vessels (100–1000L) providing optimal temperature, pH, substrate, and oxygen.
• Stirred-tank Bioreactor: A cylindrical vessel with a curved base and an agitator to ensure oxygen availability and even mixing.

Downstream Processing

• Separation and Purification: The final stages of the process before marketing.
• Formulation: Addition of suitable preservatives.
• Quality Control: Rigorous testing and clinical trials (especially for drugs/vaccines) are mandatory before public release.