Principles of Biotechnology
Biotechnology Overview:
- Involves techniques using live organisms or their enzymes to produce beneficial products and processes for human use.
- Traditional processes like curd, bread, or wine-making can be considered early forms of biotechnology.
Modern Biotechnology Focus:
- Today, biotechnology is associated with processes employing genetically modified organisms (GMOs) on a larger scale.
Diverse Biotechnological Processes:
- Encompasses a wide range of techniques:
- In vitro fertilization for ‘test-tube’ baby creation.
- Gene synthesis and utilization.
- Development of DNA vaccines.
- Correction of defective genes.
- Encompasses a wide range of techniques:
European Federation of Biotechnology (EFB) Definition:
- EFB defines biotechnology as:
- “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”
- EFB defines biotechnology as:
PRINCIPLES OF BIOTECHNOLOGY
Core Techniques in Modern Biotechnology:
Genetic Engineering:
- Objective: Alteration of genetic material (DNA and RNA).
- Implementation: Introduction into host organisms.
- Outcome: Modification of host organism’s phenotype.
Bioprocess Engineering:
- Objective: Maintain a sterile environment in chemical processes.
- Purpose: Enable the growth of specific microbe/eukaryotic cell.
- Applications: Large-scale production of biotechnological products (antibiotics, vaccines, enzymes).
Conceptual Development of Genetic Engineering:
Advantages of Sexual Reproduction:
- Allows variations and unique genetic combinations.
- Offers benefits to the organism and population.
Limitations of Traditional Hybridisation:
- Undesirable genes often accompany desired genes.
- Genetic engineering techniques overcome this limitation.
Genetic Engineering Techniques:
- Creation of recombinant DNA.
- Use of gene cloning and gene transfer.
Overcoming Genetic Limitations:
- Genetic engineering enables isolation and introduction of desirable genes only.
- Avoids inclusion of undesirable genes into the target organism.
Fate of Transferred DNA:
- Transferred DNA may not multiply in the progeny cells initially.
- Integration into the host genome enables replication.
Integration Mechanism:
- Alien DNA linked with the origin of replication in the chromosome.
- Specific DNA sequence called the origin of replication initiates replication.
Cloning or Replication:
- Alien DNA linked with the origin of replication can replicate and multiply.
- Making multiple identical copies of the template DNA.
Construction of Recombinant DNA:
- First instance: Linking a gene encoding antibiotic resistance with a native plasmid.
- Cohen and Boyer (1972) isolated the antibiotic resistance gene using restriction enzymes.
- DNA ligase linked the cut DNA with the plasmid, creating recombinant DNA.
Plasmid as a Vector:
- Plasmid acts as a vector to transfer the linked DNA into the host organism.
- Similar to a mosquito acting as a vector for transferring the malarial parasite.
Enzymatic Role:
- DNA ligase facilitates the linking of antibiotic resistance gene with the plasmid vector.
Cloning of Antibiotic Resistance Gene:
- The linked DNA replicates in E. coli, termed as cloning of antibiotic resistance gene.
Basic Steps in Genetic Modification:
- Identification of DNA with desirable genes.
- Introduction of identified DNA into the host.
- Maintenance of introduced DNA in the host and its transfer to progeny.
Construction of Recombinant DNA:
Objective: Linking a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium.
Isolation of Antibiotic Resistance Gene:
- Enzymatic Cutting: Use of restriction enzymes (molecular scissors) to cut a specific piece of DNA from a plasmid responsible for antibiotic resistance at precise locations.
- Target DNA: Antibiotic resistance gene.
Plasmid DNA as Vector:
- Role of Plasmid: Circular autonomously replicating DNA acts as a vector to transfer the antibiotic resistance gene.
- Vector Selection: Native plasmid of Salmonella typhimurium chosen as the vector.
Enzymatic Linking:
- DNA Ligase: Enzyme responsible for linking the cut antibiotic resistance gene with the plasmid DNA.
- Formation of Recombinant DNA: Binding of the gene to the plasmid, creating a new circular autonomously replicating DNA.
Transfer into Host Organism:
- Host Selection: Escherichia coli chosen as the host organism, closely related to Salmonella.
- Vector Role: Plasmid acts as a vector to deliver the recombinant DNA into the host.
Replication in Host:
- Host’s DNA Polymerase: Enzyme in E. coli used for replication.
- Cloning: Multiplication of copies of the antibiotic resistance gene within E. coli.
- Result: Cloning of the antibiotic resistance gene in E. coli achieved.
