Biotechnology: Principles and Processes - Chapter 11 Detailed Study Guide

Biotechnology: Definitions and Core Principles

• Definition of Biotechnology: Biotechnology deals with the techniques of using living organisms or enzymes from these organisms to produce products and processes useful to human beings.
• The European Federation of Biotechnology (EFB) Definition: The EFB has provided a definition that integrates both traditional and molecular perspectives: "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services."
• Core Principles of Modern Biotechnology: Two primary techniques enabled the birth of modern biotechnology:     * Genetic Engineering: A technique used to alter the chemistry of genetic material ($RNA/DNA$) to introduce it into a host organism, thereby changing the phenotype of the host organism.     * Maintenance of Sterile Conditions: Maintaining a microbial contamination-free environment in chemical engineering processes. This enables the growth of only the desired microbe or eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

Recombinant DNA (rDNA) and Its Applications

• Definition of Recombinant DNA (rDNA): rDNA is DNA from two different sources that has been combined in vitro (outside a living organism).
• Reasons for Creating rDNA:     * To create protein products.     * To create multiple copies of genes.     * To insert foreign genes into other organisms to provide them with new traits.
• Recombinant DNA Technology: This technique permits the isolation of a desired gene from any organism and its transfer and expression into an organism of choice. It is a popular term for genetic engineering.
• Applications:     * Production of transgenic organisms to obtain pharmaceutical proteins.     * Example: Human insulin produced by a transgenic E. coli strain that contains and expresses the human insulin gene.     * Through rDNA technology, large quantities of recombinant proteins with medicinal value can be produced on commercial scales.

The Historical Milestone of Stanley Cohen and Herbert Boyer

• Creation of the First Artificial rDNA: In 1972, Stanley Cohen and Herbert Boyer invented the technique of DNA cloning, allowing genes to be transplanted between different biological species.
• Process:     * They isolated an antibiotic-resistant gene from the plasmid of Salmonella typhi.     * The cutting of DNA at specific locations became possible with the discovery of "molecular scissors," known as restriction enzymes.     * They used a plasmid as a vector to deliver the alien DNA from Salmonella typhi into the host organism, E. coli.     * The antibiotic-resistant gene was linked with the E. coli plasmid using the enzyme DNA ligase.
• Results: The resulting recombinant molecule was a circular, autonomously replicating DNA created in vitro. When transferred to E. coli, the gene was stable, replicated along with the E. coli plasmid, and was transcribed and translated, expressing antibiotic resistance. This was a major breakthrough in genetic engineering.

Concepts of Cloning and Organism Modification

• Cloning: The ability to multiply copies of recombinant DNA in E. coli is known as cloning.
• Gene Cloning: The process of synthesizing multiple copies of a particular DNA sequence using a bacterial cell or another organism as a host. The gene of interest is integrated into a rDNA vector (often a plasmid) and amplified in an appropriate host cell.
• Basic Steps to Genetically Modify an Organism:     1. Identification of DNA with a desirable gene.     2. Introduction of the identified DNA into the host.     3. Maintenance of the introduced DNA in the host and transfer of the DNA to the progeny.

Tools of rDNA Technology: 1. Restriction Enzymes

• Discovery: The first restriction enzyme (RE) was discovered or isolated by Hamilton in 1970. The first RE was Hind II.
• Nature and Function:     * They are a group of enzymes that cut DNA strands, each having a characteristic base sequence at which it cleaves.     * Referred to as "molecular scissors."     * They exist in many bacteria as part of a defense mechanism called the restriction-modification system.
• The Restriction-Modification System:     1. Restriction Endonuclease: The first component, which selectively recognizes a specific DNA sequence and degrades any DNA containing that sequence.     2. Modification Enzyme: The second component, which adds a methyl group to one or two bases (usually within the recognized sequence). After methylation, the RE fails to recognize and cleave the DNA, thus protecting the bacteria's own DNA.
• Specificity: Each RE recognizes a specific host sequence called a restriction sequence. For example, Hind II specifically recognizes a particular sequence of six base pairs. To date, more than 900 REs have been isolated from over 230 strains of bacteria, each with a specific recognition sequence.

Naming and Classification of Restriction Enzymes

• Naming Convention:     * The 1st letter of the name comes from the genus.     * The 2nd and 3rd letters come from the species of the prokaryotic cell.     * A designation for the strain may follow (e.g., "R").     * Roman numerals following the name indicate the order in which they were isolated from that strain of bacteria.     * Example: EcoRI: "E" from Escherichia, "co" from coli, "R" from the strain RY13, and "I" being the first isolated.
• Classification of Nucleases:     * Exonucleases: Enzymes that digest DNA/RNA starting from the ends of the strands. They require a free end to degrade the molecule.     * Endonucleases: Enzymes that catalyze the hydrolytic cleavage of DNA in the middle of a strand or double helix. These are the critical tools for rDNA technology.

Mechanism of Restriction Enzyme and DNA Ligase

• Functioning: Each RE inspects the length of DNA for its recognition sequence. It then binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.
• Palindromic Sequences: REs recognize specific palindromic sequences of nucleotides, where the base sequence of one strand is the same as its complement when read in the opposite direction.     * Example:         * $5' \text{ GAATTC } 3'$         * $3' \text{ CTTAAG } 5'$
• Sticky Ends: REs cut the DNA strands a little away from the center of the palindromic site, but between the same two base pairs on opposite strands. This leaves single-stranded portions at the ends called "sticky ends."
• DNA Ligase: This enzyme connects the sugar-phosphate backbone of DNA. It forms the covalent bonds necessary to join DNA fragments. The presence of sticky ends facilitates the action of DNA ligase, provided the vector and source DNA were cut by the same RE.

Separation and Isolation: Gel Electrophoresis

• Concept: A technique to separate macromolecules like DNA fragments or RNA based on their size.
• Process:     1. The sample DNA is cut into fragments by restriction endonucleases.     2. DNA fragments are negatively charged due to phosphate groups.     3. When placed in an electric field, they are forced to move toward the anode through a matrix.     4. Matrix: The most common matrix is agarose gel, a natural linear polymer of D-galactose and 3,6-anhydro-L-galactose extracted from seaweeds.     5. Sieving Effect: Fragments separate by size; smaller fragments move farther through the pores of the gel.
• Visualization:     1. Fragments are stained with ethidium bromide.     2. They are exposed to UV radiation to be seen as orange-colored bands.     3. Elution: The process of cutting out the separated bands from the gel and extracting the DNA from the gel piece.
• The purified DNA fragments are then used to form rDNA by joining them with cloning vectors.

Tools of rDNA Technology: 2. Cloning Vectors

• Definition: DNA molecules that can carry a foreign DNA segment (gene of interest) into a host cell for cloning.
• Types: Plasmids (circular extrachromosomal DNA in prokaryotes like E. coli) and Bacteriophages (viruses that infect bacteria).
• Essential Features of a Vector:     * Origin of Replication (ori): The sequence responsible for initiating replication. It also controls the copy number (number of vector copies in a cell, varying from 1 to 100).     * Selectable Marker: Helps select host cells containing the vector (transformants) and eliminate non-transformants. Common markers for E. coli include resistance genes for ampicillin, chloramphenicol, tetracycline, or kanamycin.     * Cloning Sites: A vector must have at least one unique restriction endonuclease recognition site. Multiple sites for one enzyme would complicate cloning by generating several fragments.     * Size: Vectors should be small, as large molecules tend to break down during purification or manipulation.

Selection of Recombinants

• Methods:     1. Inactivation of Antibiotics: If foreign DNA is ligated at the BamHI site of the tetracycline resistance ($tet^R$) gene in the vector pBR322, the plasmid loses tetracycline resistance (insertional inactivation). However, it still possesses ampicillin resistance ($amp^R$). Transformants are selected by plating on ampicillin plates (all transformants grow), then replica plating onto tetracycline plates (only non-recombinants grow). This method is cumbersome.     2. Insertional Inactivation (Blue/White Screening): A recombinant DNA sequence is inserted within the coding sequence of the enzyme $\beta$-galactosidase. This inactivates the enzyme. In the presence of a chromogenic substrate, bacterial colonies with the inserted plasmid (recombinants) show no color, while those without the insert (non-recombinants) form blue colonies.

Vectors for Plants and Animals

• For Plants: The Ti plasmid (tumor-inducing) of Agrobacterium tumifaciens is used. A. tumifaciens is a rod-shaped, Gram-negative pathogen that causes Crown Gall disease in dicots by delivering "T-DNA" to transform plant cells. The Ti plasmid is modified to be "disarmed" (non-pathogenic) while still delivering genes.
• For Animals: Retroviruses, Adenoviruses, and Papillomaviruses have been disarmed and are used to deliver desirable genes into animal cells.

Tools of rDNA Technology: 3. Competent Host

• Concept: Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. The host must be made "competent" to accept it.
• Methods to Increase Competency:     1. Chemical Treatment: Treatment with a specific concentration of a divalent cation like Calcium ($Ca^{2+}$) increases the efficiency of DNA entry through cell wall pores.     2. Heat Shock Method: Incubating cells with rDNA on ice, followed by placing them at $42^\circ\text{C}$, and then putting them back on ice.     3. Microinjection: Recombinant DNA is directly injected into the nucleus of an animal cell using a glass micropipette.     4. Biolistics or Gene Gun: In plants, cells are bombarded with high-velocity microparticles of gold or tungsten coated with DNA.     5. Electroporation: Using electric current to create transient microscopic pores in the host cell membrane.     6. Disarmed Pathogen Vectors: Allowing modified pathogens like Agrobacterium or Retroviruses to infect the cell and transfer the rDNA.

Processes of Recombinant DNA Technology

1. Isolation of Genetic Material (DNA): Breaking the cell wall/membrane using enzymes like lysozyme (bacteria), cellulase (plants), or chitinase (fungi). RNA is removed by ribonuclease; proteins are removed by protease. Purified DNA is precipitated by adding chilled ethanol.
2. Cutting of DNA at Specific Locations: Purified DNA and vector DNA are incubated with specific REs at optimum conditions. Progression is checked via agarose gel electrophoresis.
3. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction): A process of repetitive bidirectional synthesis of DNA in vitro.     * Enzyme: Taq polymerase, a heat-resistant DNA polymerase extracted from the bacterium Thermus aquaticus found in hot springs.     * Steps of PCR:         * Denaturation: Double-stranded DNA is heated to $95^\circ\text{C}$ for 15 seconds to separate strands.         * Annealing: Two sets of primers are added to the 3' end of each separated strand for initiation.         * Extension: Taq polymerase adds complementary nucleotides to the primers. Steps are repeated up to 30 cycles to amplify DNA up to a billion times.
4. Ligation of DNA Fragment into a Vector: Vector and source DNA (cut with the same RE) are mixed with DNA ligase to form rDNA.
5. Insertion of rDNA into Host: Transformation of competent cells. Using ampicillin resistance as a selectable marker, only transformants will grow on ampicillin-containing agar plates.
6. Culturing Host Cells: Transformed cells are grown in nutrient medium at optimal conditions to allow the DNA to multiply and express the desired product.
7. Extraction of Desired Gene Product: The protein produced is called a recombinant protein.     * Small Scale: Lab-based extraction and purification.     * Large Scale: Using continuous culture systems where fresh medium is added from one side while cells and product are removed from the other to maintain the exponential growth phase.
8. Downstream Processing: Processes applied to a product before marketing, including separation of products from the reactor, purification, formulation with preservatives, and strict quality control/clinical trials.

Bioreactors

• Description: Large vessels ($100$ to $1000$ litres) in which raw materials are biologically converted into specific products. They provide optimal conditions for growth (temperature, $O_2$, pH, substrate, salts, vitamins).
• Stirred Tank Bioreactors: Commonly used, cylindrical with a curved base for mixing.
• Components:     * Agitator system     * $O_2$ delivery system     * Foam control system     * Temperature and pH control systems     * Sampling ports to withdraw culture periodically.
• Sparged Stirred Tank Reactor: A type of stirred reactor where sterile air is bubbled through to increase oxygen transfer area dramatically.