Unit 4: Biotechnology and Its Applications - Chapter 1: Principles and Processes

4.1 Principles and Processes of Biotechnology

Definition and Principles

Biotechnology is the use of living organisms or their products to create or modify products, improve plants or animals, or develop microorganisms for specific uses. 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."

Core Principles of Modern Biotechnology:

1. Genetic Engineering: The technique 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 the growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

Genetic Engineering: Recombinant DNA (rDNA) Technology

Genetic Engineering involves the manipulation of genetic material to achieve desired traits. Recombinant DNA (rDNA) Technology is a key component of genetic engineering, involving the creation of new DNA molecules by combining genetic material from different sources.

Key Components of rDNA Technology:

• Restriction Enzymes: Molecular scissors that cut DNA at specific recognition sequences.
• Cloning Vector: A DNA molecule that can carry a foreign DNA segment and replicate independently in a host cell.
• Desired Gene (Foreign DNA/Insert DNA): The gene of interest to be transferred.
• Host Organism: The organism into which the recombinant DNA is introduced for replication and expression.

Steps of Recombinant DNA Technology

1. Isolation of the Genetic Material (DNA):

   • Cells are treated with enzymes (e.g., lysozyme for bacteria, cellulase for plant cells, chitinase for fungi) to break open the cell and release DNA.
   • RNA, proteins, polysaccharides, and lipids are removed by appropriate treatments (e.g., ribonuclease for RNA, protease for proteins).
   • Purified DNA is precipitated by adding chilled ethanol.

2. Cutting of DNA at Specific Locations (Gene of Interest):

   • Restriction Enzymes (Restriction Endonucleases): These enzymes recognize specific palindromic nucleotide sequences in the DNA and cut both strands of the DNA at or near these sites.
     • EcoRI: Recognizes GAATTC and cuts between G and A, producing sticky ends.
     • HindII: The first restriction endonuclease isolated, always cuts DNA molecules at a particular point by recognizing a specific sequence of 6 base pairs.
   • The foreign DNA (gene of interest) and the vector DNA are cut with the same restriction enzyme to produce compatible sticky ends.

3. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction):

   • PCR is a technique used to synthesize multiple copies of the gene of interest in vitro.
   • Steps:
     • Denaturation: Heating the DNA to separate the two strands.
     • Annealing: Primers (short, chemically synthesized oligonucleotides) bind to the separated DNA strands.
     • Extension: Taq polymerase (a thermostable DNA polymerase) synthesizes new DNA strands using the primers and the template DNA.
   • Repeated cycles lead to exponential amplification of the DNA segment.

4. Ligation of DNA Fragments into a Vector:

   • The cut gene of interest and the cut vector DNA are mixed.
   • DNA Ligase: This enzyme joins the sticky ends of the foreign DNA and the vector DNA, forming a recombinant DNA molecule (rDNA).

5. Insertion of Recombinant DNA into the Host Cell/Organism:

   • The recombinant DNA is introduced into a suitable host cell (e.g., E. coli).
   • Methods of Gene Transfer (Transformation):
     • Heat Shock Method: Bacterial cells are made competent by treating them with a specific concentration of a divalent cation (e.g., calcium chloride), which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. The cells are then incubated with recombinant DNA on ice, followed by a brief heat shock (42°C), and then placed back on ice. This enables the bacteria to take up the rDNA.
     • Microinjection: Recombinant DNA is directly injected into the nucleus of an animal cell.
     • Biolistics (Gene Gun): Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. Suitable for plant cells.
     • Disarmed Pathogen Vectors: Using disarmed (non-pathogenic) pathogens (e.g., Agrobacterium tumefaciens for plants, retroviruses for animals) to deliver the gene of interest.

6. Selection and Screening of Transformed Host Cells:

   • Only a small percentage of host cells take up the recombinant DNA. Methods are used to identify these transformed cells.
   • Antibiotic Resistance Markers: Vectors often contain genes conferring resistance to antibiotics (e.g., ampicillin, tetracycline). Transformed cells will grow on media containing the antibiotic, while non-transformed cells will die.
   • Blue-White Screening: A method used to detect the presence of recombinant plasmids. The vector contains the lacZ gene (encoding β-galactosidase). If foreign DNA is inserted into lacZ, it inactivates the gene (insertional inactivation). When grown on a medium containing X-gal (a chromogenic substrate), non-recombinant colonies (with intact lacZ) will be blue, while recombinant colonies (with inactivated lacZ) will be white.

7. Obtaining the Foreign Gene Product (Cloning and Expression):

   • Once the recombinant DNA is in the host cell, the goal is to express the foreign gene to produce the desired protein.
   • Cloning: The process of making multiple identical copies of a DNA fragment or an entire organism.
   • Optimized Expression: Conditions (e.g., nutrient media, temperature, pH) are optimized for the host cell to produce the desired protein in large quantities.

Bioreactors: Stirred Tank, Sparged Tank

Bioreactors are large vessels (100-1000 liters) used for large-scale production of biotechnological products. They provide optimal conditions for the growth of desired microorganisms or cells and for the synthesis of the product.

1. Stirred-Tank Bioreactors:

   • Most commonly used type.
   • Features:
     • Stirrer System: Ensures uniform mixing of contents (reactants, products, cells) and availability of oxygen throughout the reactor.
     • Oxygen Delivery System: Provides oxygen for aerobic processes.
     • Foam Control System: Prevents foam build-up.
     • Temperature Control System: Maintains optimal temperature.
     • pH Control System: Maintains optimal pH.
     • Sampling Ports: Allows withdrawal of small volumes of culture periodically for testing.
   • Types: Simple stirred-tank bioreactor, sparged stirred-tank bioreactor.

2. Sparged Stirred-Tank Bioreactors:

   • Similar to stirred-tank bioreactors, but air (or gas) is sparged (bubbled) through the reactor, increasing the surface area for oxygen transfer.
   • The stirring system helps in mixing and oxygen distribution.

Downstream Processing

Downstream processing refers to the processes involved in the separation and purification of the desired product (e.g., protein, enzyme, antibiotic) from the bioreactor culture. It is a critical step after the biosynthetic stage.

Steps in Downstream Processing:

1. Separation: The product is separated from the cell culture (e.g., by centrifugation, filtration).
2. Purification: The separated product is purified using various techniques (e.g., chromatography, electrophoresis, precipitation).
3. Formulation: The purified product is formulated with suitable preservatives.
4. Quality Control Testing: Rigorous testing is performed to ensure the purity, safety, and efficacy of the product.
5. Clinical Trials (for drugs): If the product is a drug, it undergoes clinical trials.

Downstream processing and quality control testing are crucial for ensuring that the final product is fit for marketing. Each product undergoes thorough quality control testing. The downstream processing varies from product to product.