Principles of Biotechnology
Modern biotechnology is based on two key principles:
1. Genetic Engineering: This involves modifying the DNA of a target organism
to change its phenotype. It allows for the manipulation of genetic material to
produce desired traits in the organism.
2. Bioprocess Engineering: This principle focuses on maintaining sterile
conditions to cultivate large quantities of microorganisms and eukaryotic cells.
These are essential for producing biotechnological products such as
antibiotics, enzymes, and vaccines.
The techniques of genetic engineering typically follow these steps:
Isolating the desired DNA fragment from the donor organism.
Inserting this DNA into a vector.
Transferring the vector into a suitable host organism.
Cloning the recombinant DNA within the host to replicate and express the
desired trait.
What Is Recombinant DNA Technology?
Recombinant DNA technology, also known as genetic engineering, is the process of combining
DNA molecules from two different organisms to create recombinant DNA. This technique
allows scientists to introduce new genetic material into an organism to produce desired traits
or products.
The steps involved in recombinant DNA technology are:
1. Isolation of DNA: The DNA of interest is extracted from the donor organism.
2. DNA Fragmentation: Restriction endonucleases are used to cut the DNA into
fragments.
3. Ligation: The desired DNA fragment is inserted into a vector (carrier DNA).
4. Transfer: The recombinant DNA is introduced into a suitable host organism.
5. Culture: The host cells containing recombinant DNA are grown in a nutrient
medium.
6. Extraction: The desired product, such as a protein or enzyme, is extracted from the
cultured cells.
DNA Cloning
DNA cloning is the process of producing multiple identical copies of a specific DNA fragment.
This is an essential technique in genetic engineering and biotechnology. To achieve DNA
cloning, cloning vectors are used, and these vectors must possess the following properties:
1. Small Size but Large DNA Capacity: The vector should be small for easier
manipulation but must carry a large DNA insert.
2. Origin of Replication: It should have an origin of replication to enable autonomous
replication within the host organism.
3. Restriction Site: The vector must have a restriction site where the DNA fragment can
be inserted.
4. Selectable Marker: It should include a marker gene to help identify and select
recombinant organisms.
5. Multiple Cloning Sites: The vector should have several sites where different DNA
fragments can be inserted for versatility in cloning experiments.
What Is Bioprocess Engineering?
Bioprocess engineering involves the cultivation and multiplication of cells in bioreactors to
produce large quantities of biological products. This process results in a high yield of the
desired proteins or other biological materials. Once produced, the products
undergo downstream processing, which includes purification and quality control checks.
Before being released or further developed, the products are tested to ensure safety and
effectiveness. Bioprocess engineering is widely used in the production of antibiotics, vaccines
and other therapeutic medicines playing a important role in the biotechnology industry.
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.
