Biotechnology Principles and Processes Biology Notes - rDNA Technology, Tools, Restriction Enzymes, Vectors, PCR and Bioreactors

• Biotechnology is the technique of using live organisms or their enzymes for products and processes useful to humans.
• The European Federation of Biotechnology (EFB) defines biotechnology as “the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services”.

Biotechnology deals with:

• Microbe-mediated processes (e.g., making curd, bread, wine).
• In vitro fertilization (test-tube baby programme).
• Synthesis and use of a gene.
• Preparation of DNA vaccines.
• Correcting a defective gene.

Principles of Biotechnology

Core Techniques of Modern Biotechnology

• Genetic Engineering: The technique in which genetic material (DNA and RNA) is chemically altered and introduced into host organisms to change the phenotype.
• Bioprocess Engineering: Maintenance of a sterile environment in chemical engineering processes for growing desired microbes or eukaryotic cells for the manufacture of antibiotics, vaccines, enzymes, etc.

Basic Steps in Genetically Modifying an Organism

1. Identification of DNA with Desirable Genes: Traditional hybridization leads to inclusion and multiplication of undesirable genes along with desired genes. In genetic engineering, only desirable genes are introduced.
2. Introduction of the Identified DNA into the Host: A vector DNA, such as a plasmid, is used to deliver an alien piece of DNA into the host organism.
3. Maintenance of Introduced DNA in the Host and Transfer to Its Progeny: A piece of alien DNA lacks the sequence called origin of replication (ori) needed for starting replication, so it cannot multiply itself in the progeny cells. Hence, alien DNA is integrated into the recipient genome (which has ori). It multiplies and is inherited along with host DNA.

• The process of joining and inserting a foreign piece of DNA into a host organism to produce new genetic combinations is called recombinant DNA technology.
• The first recombinant DNA (rDNA) was produced by Stanley Cohen and Herbert Boyer in 1972.
• They isolated an antibiotic resistance gene from a plasmid of Salmonella typhimurium, linked it with a plasmid vector, and transferred it into Escherichia coli. As a result, the gene was expressed and multiplied in E. coli.

Tools of Recombinant DNA Technology

Restriction Enzymes (‘Molecular Scissors’)

• Enzymes that cut DNA at specific sites into fragments.
• They belong to a class of enzymes called nucleases.
• In 1963, two enzymes restricting bacteriophage growth in Escherichia coli were isolated:
  • One added methyl groups to DNA.
  • The other, a restriction endonuclease, cut DNA.
• More than 900 restriction enzymes have been isolated from over 230 bacterial strains.

Naming of the Restriction Enzymes

• The first letter indicates the genus, and the next two letters indicate the species of the prokaryotic cell from which they were isolated.
• Example: EcoRI comes from E. coli RY13, where R denotes the strain, and Roman numerals indicate the order of isolation.

Types of Restriction Enzymes

• Exonucleases: Remove nucleotides from the ends of DNA.
• Endonucleases:
  • Cut at specific positions within the DNA (e.g., EcoRI).
  • Bind to specific recognition sequences and cut both strands at specific points.
  • The first restriction endonuclease, Hind II, recognizes a specific 6-base-pair sequence, called the recognition sequence.
  • Recognize palindromic nucleotide sequences, which read the same in the 5'→3' and 3'→5' directions.

Example: Palindromic nucleotide sequence for EcoRI:

5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'

• Restriction enzymes cut strands slightly away from the palindrome’s center, between the same bases on opposite strands, creating sticky ends that form hydrogen bonds with complementary counterparts, facilitating DNA ligase action.
• When cut by the same restriction enzyme, resultant DNA fragments have identical sticky ends, joined by DNA ligases.

Cloning Vector

• A DNA molecule that carries a foreign DNA segment and replicates inside host cells (e.g., plasmids, bacteriophages).
• Plasmids: Autonomously replicating circular extra-chromosomal DNA in bacteria, with 1–2 or 15–100 copies per cell.
• Bacteriophages: Have high genome copy numbers within bacterial cells.
• Cloning vectors multiply the linked DNA to the vector’s copy number in the host.

Features Required for Cloning into a Vector

a. Origin of Replication (ori):

• Sequence where replication starts.
• DNA linked to ori replicates within host cells, controlling copy number. High-copy-number vectors are used for multiple target DNA copies.

b. Selectable Marker (Marker Gene):

• Genes that select transformants and eliminate non-transformants.
• Transformation: Introduction of DNA into a host bacterium. Transformants have the plasmid; non-transformants do not.
• In E. coli, selectable markers include antibiotic resistance genes (e.g., ampicillin, chloramphenicol, tetracycline, kanamycin). Normal E. coli lacks such resistance.

c. Cloning Sites:

• Recognition sites for restriction enzymes.
• Vectors need single or few recognition sites to link alien DNA.
• Multiple recognition sites generate several fragments, complicating cloning.
• Ligation occurs at a restriction site within an antibiotic resistance gene (e.g., in pBR322, foreign DNA is ligated at the BamHI site of the tetracycline resistance gene, forming a recombinant plasmid).

• Restriction sites: Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I.
• ori
• Antibiotic resistance genes: ampR and tetR.
• Rop: codes for the proteins involved in the replication of plasmid.

d. Vectors for Cloning Genes in Plants & Animals:

• Agrobacterium tumefaciens: Delivers T-DNA to transform plant cells into tumors, producing pathogen-required chemicals. Its tumor-inducing (Ti) plasmid is modified into a non-pathogenic cloning vector for plant gene delivery.
• Retroviruses: Transform animal cells into cancerous cells, used to deliver desirable genes into animal cells.

• Insertional Inactivation: Inserting foreign DNA into a bacterial gene inactivates it (e.g., recombinant plasmids lose tetracycline resistance).
• After introducing plasmids into E. coli, three cell types result:
  • Non-transformants: No plasmid, not resistant to tetracycline or ampicillin.
  • Transformants with non-recombinant plasmid: Resistant to both tetracycline and ampicillin.
  • Transformants with recombinant plasmid: Resistant only to ampicillin.
• Recombinants are selected by plating transformants on ampicillin medium, then transferring to tetracycline medium. Recombinants grow on ampicillin but not tetracycline; non-recombinants grow on both.
• One antibiotic resistance gene selects transformants; the inactivated gene selects recombinants.
• This selection is complex, requiring two plates. Alternative selectable markers use chromogenic substrates:
  • Recombinant DNA inserted into the β-galactosidase gene inactivates it, producing colorless colonies (recombinants).
  • Non-recombinant plasmids produce blue colonies with chromogenic substrates.

Competent Host (For Transformation with Recombinant DNA)

• Since DNA is hydrophilic, it cannot pass through cell membranes. Bacterial cells are made competent to take up alien DNA or plasmids.
• Process:
  • Treat cells with a divalent cation (e.g., calcium) to create pores in the cell wall.
  • Incubate with recombinant DNA on ice, apply heat shock at 42°C, then return to ice to enable DNA uptake.

Other Methods to Introduce Alien DNA into Host Cells

• Micro-injection: Recombinant DNA is injected directly into an animal cell’s nucleus.
• Biolistics (Gene Gun): Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA, suitable for plants.
• ‘Disarmed Pathogen’ Vectors: Pathogens like A. tumefaciens infect cells and transfer recombinant DNA into the host.

Processes of Recombinant DNA Technology

1. Isolation of the Genetic Material (DNA)

• Treat bacterial cells, plant, or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plants), or chitinase (fungus) to break the cell, releasing DNA and other macromolecules (RNA, proteins, polysaccharides, lipids).
• Remove RNA with ribonuclease, proteins with protease, and other molecules with appropriate treatments.
• Add chilled ethanol to precipitate purified DNA as fine threads in the suspension.

2. Cutting of DNA at Specific Locations

• Incubate purified DNA with a restriction enzyme to produce DNA digests, separated by gel electrophoresis.
• Agarose gel electrophoresis checks restriction enzyme digestion progress. DNA, being negatively charged, moves toward the anode, with smaller fragments moving farther due to the sieving effect of agarose gel (a polymer from seaweed).
• Repeat the process with vector DNA.
• Stain DNA fragments with ethidium bromide to visualize as bright orange bands under UV radiation.
• Cut DNA bands from the agarose gel (elution), mix the gene of interest and cut vector, and add ligase to create recombinant DNA.

3. Amplification of Gene of Interest using PCR

• Polymerase Chain Reaction (PCR) synthesizes multiple copies of the gene of interest in vitro using two sets of primers and the enzyme DNA polymerase.
• Primers are small, chemically synthesized oligonucleotides complementary to DNA regions.

Steps of PCR

• Denaturation: Heat target DNA at 94°C to separate strands, each acting as a template for DNA synthesis.
• Annealing: Join two primers at 52°C to the 3’ end of DNA templates.
• Extension: Add nucleotides to the primer using thermostable Taq polymerase, isolated from Thermus aquaticus, which remains active at high temperatures during denaturation.

• Continuous replication amplifies the DNA segment up to 1 billion copies.
• The amplified fragment can be ligated with a vector for further cloning.

4. Insertion of Recombinant DNA into Host Cell

• Introduce ligated DNA into a recipient (host) cell/organism, which takes up DNA from its surroundings.
• If recombinant DNA with an ampicillin-resistant gene is transferred into E. coli cells, the host becomes ampicillin-resistant.
• Spread transformed cells on agar plates containing ampicillin; only transformants grow, while untransformed cells die.

5. Obtaining the Foreign Gene Product

• The aim is to produce a desirable protein.
• A protein-encoding foreign gene expressed in a heterologous host is called a recombinant protein.
• Grow cells with foreign genes in the laboratory, extract the desired protein, and purify it using separation techniques.
• Use a continuous culture system to multiply cells, where used medium is drained and fresh medium added, maintaining physiologically active cells for larger biomass and more protein.

Bioreactors

• Vessels where raw materials are biologically converted into specific products (e.g., enzymes) using microbial, plant, animal, or human cells.
• Produce large quantities, processing 100–1000 liters of culture.
• Provide optimal growth conditions (pH, temperature, substrate, salts, vitamins, oxygen).
• Commonly used are stirred-tank bioreactors, cylindrical or with a curved base, with a stirrer or air bubbling for mixing and oxygen availability.
• Components include:
  • An agitator system.
  • An oxygen delivery system.
  • A foam control system.
  • A temperature control system.
  • A pH control system.
  • Sampling ports for periodic culture withdrawal.

6. Downstream Processing

• A series of processes for separation and purification of products after the biosynthetic stage.
• The product is formulated with suitable preservatives, undergoes clinical trials, and strict quality control testing.