CBSE Class 12 Biology Notes Chapter 11 Biotechnology Principles and Processes

Cloning Vector

Cloning vectors are essential tools in recombinant DNA technology, allowing for the insertion and replication of foreign DNA in a host organism. Plasmids and bacteriophages are commonly used vectors. They have the ability to replicate independently within bacterial cells making them ideal for cloning purposes. Bacteriophages, due to their high copy numbers, ensure numerous copies of their genome within bacterial cells.

Key features of cloning vectors include:

1. Origin of Replication (ori) : A sequence from which replication begins. When DNA is linked to this sequence, it can replicate within host cells, controlling the copy number of the linked DNA.
2. Selectable Marker : This helps identify and select transformed cells by encoding resistance to antibiotics such as ampicillin or tetracycline. These markers are used to differentiate between non-transformants and transformants, ensuring only cells with recombinant DNA survive.
3. Cloning Sites : Single recognition sites for restriction enzymes are required for the insertion of foreign DNA. Multiple restriction sites could generate fragments that complicate the cloning process. The insertion of foreign DNA typically disrupts one of the antibiotic resistance genes, helping identify successful recombinants.
4. Insertional Inactivation : This technique is used to identify recombinant plasmids. When a foreign DNA fragment is inserted, it disrupts the coding sequence of a gene, such as the lac Z gene in the Blue-White selection process. Recombinant colonies appear white due to the inactivation of the lac Z gene, while non-recombinants appear blue.
5. Vectors for Plants and Animals : In plants, the bacterium Agrobacterium tumefaciens delivers T-DNA, transforming plant cells and modifying them into tumor cells. The Ti plasmid of Agrobacterium has been modified to serve as a cloning vector, eliminating its pathogenic properties. In animals, retroviruses are used as vectors to insert recombinant DNA and transform cells, such as inducing cancerous transformations.

Transformation of Host Cells

Transformation of recombinant DNA into host cells can be achieved using various methods, including:

1. Chemical Treatment : Using divalent calcium ions, bacterial cell walls become more permeable to take up recombinant plasmids.
2. Heat Shock : Incubating the host cells on ice, followed by a brief exposure to 42°C, facilitates the uptake of recombinant DNA.
3. Microinjection : In this method, recombinant DNA is directly injected into the host cell's nucleus using a glass micropipette.
4. Biolistics/Gene Gun Method : Primarily used for plant cells, microscopic particles of gold or tungsten coated with DNA are bombarded into the host cells.
5. Disarmed Pathogens : Agrobacterium tumefaciens , when allowed to infect cells, transfers recombinant DNA into the host.

Processes of Recombinant DNA Technology

Recombinant DNA technology involves several key steps:

1. Isolation of DNA : The genetic material is isolated from cells using enzymes such as lysozyme (for bacteria), cellulase (for plant cells), and chitinase (for fungi).
2. Fragmentation : Restriction endonucleases cut the DNA at specific sites, and agarose gel electrophoresis is used to monitor the progress.
3. Amplification of DNA (PCR) : The polymerase chain reaction (PCR) technique is used to generate multiple copies of the gene of interest. PCR involves repeated cycles of denaturation, primer annealing, and extension, utilizing thermostable DNA polymerase from Thermus aquaticus .
4. Insertion of Recombinant DNA : The host cells are made competent to take up the recombinant DNA, often through chemical treatments or heat shock.
5. Gene Expression : The foreign DNA multiplies in the host cell and expresses the desired protein. Optimal conditions are maintained in bioreactors to ensure the production of the recombinant protein.
6. Downstream Processing : Once the desired product is obtained, it undergoes purification and quality control before being prepared for the market. In the case of drugs, clinical trials are conducted before public release.

Benefits of CBSE Class 12 Biology Notes Chapter 11

• Understanding Core Concepts : Grasping the fundamental principles of biotechnology helps in understanding how biological systems can be manipulated for various applications essential for exam questions on theory and applications.
• Application of Knowledge : Knowledge of genetic engineering and bioprocess engineering allows students to relate theoretical concepts to real-world scenarios making it easier to answer application-based questions.
• Enhanced Problem-Solving Skills : Studying the techniques and processes of recombinant DNA technology fosters analytical thinking and problem-solving skills crucial for tackling complex exam problems.
• Current Relevance : Biotechnology is a rapidly evolving field with significant implications in medicine, agriculture and industry.
• Preparation for Future Studies : A solid foundation in biotechnology prepares students for advanced studies or careers in biomedical research, genetic engineering and pharmaceutical development, setting the stage for future academic and professional success.
• Boosting Confidence : A thorough grasp of the subject matter boosts confidence in answering exam questions leading to better performance and reduced anxiety.