Biotechnology Principles And Processes Class 12 Biology Chapter 9 Notes

Biotechnology Principles and Processes Class 12 Biology Chapter 9 Notes

Principles of Biotechnology

Genetic Engineering

Techniques to alter the chemistry of genetic material (DNA and RNA).
Introducing modified genetic material into host organisms to change the host organism’s phenotype.

Bioprocess Engineering

Ensuring a sterile, microbial contamination-free environment in chemical engineering processes.
Enabling the growth of desired microorganisms or eukaryotic cells for large-scale production of biotechnological products like antibiotics, vaccines, enzymes, etc.

Conceptual Development of Genetic Engineering

Sexual reproduction provides genetic variation, while asexual reproduction preserves genetic information.
Traditional hybridization often introduces undesirable genes along with desired ones.
Genetic engineering techniques, including recombinant DNA creation, gene cloning, and gene transfer, enable the isolation and introduction of only desirable genes without unwanted genetic material.

Fate of Transferred DNA in Alien Organisms

Alien DNA transferred to an organism may not replicate in progeny cells.
Integration into the recipient organism’s genome enables multiplication, as it becomes part of a replicating chromosome with an origin of replication.
Alien DNA linked with the origin of replication can replicate and multiply itself in the host organism, a process akin to cloning.

Construction of Recombinant DNA

The first recombinant DNA was constructed by linking an antibiotic resistance gene with a native plasmid of Salmonella typhimurium.
Stanley Cohen and Herbert Boyer achieved this in 1972 using restriction enzymes to cut the DNA and DNA ligase to join it with the plasmid.
Plasmids act as vectors to transfer the DNA.
The linked antibiotic resistance gene and plasmid become recombinant DNA, which can replicate in a host organism such as Escherichia coli.

Genetic Modification Steps

Identification of DNA with Desirable Genes
- Locate and select DNA containing desired genes.
Introduction of Identified DNA into the Host
- Transfer the selected DNA into the host organism.
Maintenance of Introduced DNA in the Host and Transfer to Progeny
- Ensure the introduced DNA is maintained and passed on to the offspring of the host organism.

These principles and techniques underlie the field of biotechnology, enabling the manipulation and modification of organisms for various applications in medicine, agriculture, and industry.

Tools of Recombinant DNA Technology Notes

Recombinant DNA technology is a powerful tool in biotechnology, allowing scientists to manipulate and modify DNA for various applications. Several key tools are essential for the success of this technology:

1. Restriction Enzymes in Recombinant DNA Technology

In 1963, two enzymes affecting bacteriophage growth in Escherichia coli were discovered. One added methyl groups to DNA, while the other was named restriction endonuclease, which cut DNA.
Hind II, the first restriction endonuclease, was isolated in 1968. It recognized a specific DNA nucleotide sequence, known as the recognition sequence for Hind II.
Over 900 restriction enzymes from various bacterial strains have been identified, each recognizing different recognition sequences.
These enzymes are named based on the genus and species of the prokaryotic cell they were isolated from (e.g., EcoRI from Escherichia coli RY 13), with Roman numerals indicating the order of isolation.

Types of Nucleases

Nucleases are enzymes that break down nucleic acids. There are two kinds: exonucleases and endonucleases.
Exonucleases remove nucleotides from the ends of DNA, while endonucleases make cuts at specific positions within DNA.

Function of Restriction Endonucleases

Restriction endonucleases recognize specific sequences within DNA, known as recognition sequences.
These enzymes cut both strands of the DNA double helix at specific points within their sugar-phosphate backbones.
Recognition sequences are palindromic, meaning they read the same forward and backward on both DNA strands (e.g., 5′ GAATTC 3′ and 3′ CTTAAG 5′).
Restriction enzymes cut the DNA strands slightly away from the center of the palindrome sites, leaving single-stranded portions with sticky ends that can form hydrogen bonds with complementary DNA ends.

Applications in Genetic Engineering

Restriction endonucleases are used in genetic engineering to create recombinant DNA molecules, combining DNA from different sources.
DNA fragments cut with the same restriction enzyme have matching sticky ends and can be joined together using DNA ligases.
For successful recombinant DNA formation, both the vector and the source DNA must be cut with the same restriction enzyme.

Separation and Isolation of DNA Fragments

DNA fragments resulting from restriction enzyme digestion can be separated by gel electrophoresis, a technique that uses an electric field to force DNA fragments to move through a gel matrix.
Agarose gel is commonly used for this purpose, and DNA fragments separate based on size, with smaller fragments traveling farther.
The separated DNA fragments are visualized by staining with ethidium bromide and exposing the gel to UV radiation.
After separation, DNA fragments are eluted from the gel, purified, and used for constructing recombinant DNA with cloning vectors.

2. Cloning Vectors in Genetic Engineering

Cloning vectors are essential tools in genetic engineering, enabling the replication and manipulation of foreign DNA within host cells.
Plasmids and bacteriophages possess the ability to replicate independently within bacterial cells, making them suitable vectors for gene cloning.
By linking foreign DNA with plasmid or bacteriophage DNA, the foreign DNA can be replicated within host cells, multiplying its copy number.

Features Required for Cloning Vectors

Origin of Replication (ori)
- This sequence serves as the starting point for DNA replication.
- Any DNA fragment linked to this sequence can replicate within host cells.
- The origin of replication also controls the copy number of the linked DNA.
- High copy number origins are preferred for obtaining numerous copies of the target DNA.
Selectable Marker
- Vectors require a selectable marker in addition to the origin of replication.
- A selectable marker aids in distinguishing and eliminating non-transformants while allowing the selective growth of transformants.
- Common selectable markers include antibiotic resistance genes (e.g., ampicillin, chloramphenicol, tetracycline, or kanamycin) that are not naturally present in the host organism.
- Transformants carrying the vector with the selectable marker gene can grow in the presence of the corresponding antibiotic.
Cloning Sites
- Vectors should have few recognition sites for commonly used restriction enzymes to facilitate the insertion of foreign DNA.
- Multiple recognition sites within the vector can lead to the generation of various fragments, complicating gene cloning.
- Ligation of foreign DNA typically occurs at a restriction site present in one of the antibiotic resistance genes.
- Insertion of foreign DNA disrupts one antibiotic resistance gene, allowing for selection of recombinants.
Alternative Selectable Markers
- Cloning vectors may employ alternative selectable markers based on the ability to produce color in the presence of a chromogenic substrate.
- Insertional inactivation of a gene (e.g., β-galactosidase) within the vector occurs upon insertion of foreign DNA.
- Colonies containing recombinant vectors with inserted DNA do not produce color with the chromogenic substrate, distinguishing them from non-recombinant colonies.

Vectors for Cloning in Plants and Animals

Lessons from bacteria and viruses have guided the development of vectors for gene transfer into plants and animals.
Agrobacterium tumefaciens, a plant pathogen, delivers a piece of DNA known as “T-DNA” to transform plant cells, directing them to produce chemicals required by the pathogen.
Retroviruses in animals can transform normal cells into cancerous cells.
Modified versions of these pathogens have been used as vectors to deliver desired genes into plants and animal cells.
These vectors, derived from pathogens, have been disarmed and repurposed for safe and efficient gene delivery into eukaryotic hosts.

3. Making Bacterial Cells Competent for DNA Uptake

DNA is a hydrophilic (water-attracting) molecule and cannot pass through the hydrophobic (water-repelling) lipid bilayer of cell membranes.
To introduce plasmid DNA or other recombinant DNA into bacterial cells, the cells must first be made “competent,” meaning they are primed to efficiently take up foreign DNA.
This is achieved by treating the bacterial cells with specific conditions that enhance their ability to absorb DNA.

Making Bacterial Cells Competent

One common method for making bacterial cells competent involves the treatment of cells with divalent cations, such as calcium ions (Ca2+).
The addition of calcium ions alters the properties of the cell membrane and cell wall, making them more permeable to DNA molecules.
As a result, the efficiency of DNA entry into the bacterial cells is significantly increased.

Forced Uptake of Recombinant DNA

Once bacterial cells are made competent, recombinant DNA can be introduced into them.
This is typically achieved by incubating the competent cells with the recombinant DNA on ice, followed by a brief heat shock at 42°C.
The heat shock is crucial as it helps the DNA to enter the bacterium through pores in its cell wall.
After the heat shock, the cells are returned to ice to stabilize and allow them to recover.

Other Methods of DNA Introduction

There are alternative methods for introducing foreign DNA into host cells:
1. Micro-injection: In this method, recombinant DNA is directly injected into the nucleus of an animal cell.
2. Biolistics or Gene Gun: Suitable for plants, this method involves bombarding plant cells with high-velocity micro-particles (usually made of gold or tungsten) coated with DNA.
3. Disarmed Pathogen Vectors: Certain vectors derived from pathogens (e.g., Agrobacterium tumefaciens for plants) can transfer recombinant DNA into host cells when allowed to infect the cells.

Significance of Competent Hosts

Making bacterial cells competent is a critical step in genetic engineering and recombinant DNA technology.
It allows for the successful introduction of foreign DNA into host cells, facilitating various biotechnological applications.

Processes of Recombinant DNA Technology

Recombinant DNA technology encompasses several crucial steps performed in a specific sequence to manipulate and clone genes. Below are the key processes involved in recombinant DNA technology, outlined in detail:

1. Isolation of Genetic Material (DNA) in Recombinant DNA Technology

Genetic material in all organisms is nucleic acid, primarily deoxyribonucleic acid (DNA).
To work with DNA, it must be isolated in its pure form, free from other macromolecules like RNA, proteins, polysaccharides, and lipids.
DNA is enclosed within membranes or associated with other molecules in cells, necessitating the breaking of cells to release it.

Cell Disruption

To release DNA, bacterial cells, plant cells, or animal tissues need to be broken open. This is achieved through the use of enzymes:
- Lysozyme: Used to break open bacterial cells.
- Cellulase: Employed for breaking plant cell walls.
- Chitinase: Used in the case of fungal cells.
These enzymes break down the cell wall or cell membrane, releasing the cellular contents, including DNA.

Removal of RNA and Proteins

Once the cellular contents are released, they contain various molecules, including RNA and proteins.
Ribonuclease: Used to selectively degrade RNA molecules, leaving the DNA intact.
Protease: Employed to break down proteins, separating them from the DNA.
Other molecules, such as polysaccharides and lipids, may also be present and can be removed through appropriate treatments.

Precipitation of DNA

After the removal of unwanted molecules, the DNA can be further purified.
DNA can be precipitated out of the solution by adding chilled ethanol.
Precipitated DNA appears as fine threads or strands in the suspension.

2. Cutting of DNA at Specific Locations (Restriction Enzyme Digestion) in Recombinant DNA Technology

Restriction enzyme digestion is a fundamental step in recombinant DNA technology, allowing the precise cutting of DNA molecules at specific locations.
The process involves incubating purified DNA with a restriction enzyme under optimal conditions for that particular enzyme.

Monitoring Digestion with Agarose Gel Electrophoresis

To check the progress of a restriction enzyme digestion, agarose gel electrophoresis is commonly used.
DNA is a negatively charged molecule, so it moves toward the positive electrode (anode) during electrophoresis.
By comparing the sizes of DNA fragments generated in the digestion with known molecular weight markers, researchers can assess the success of the digestion and the sizes of the resulting fragments.
This step ensures that the DNA has been cut at the desired locations.

Cutting of Source DNA and Vector DNA

Both the source DNA (containing the gene of interest) and the vector DNA are subjected to restriction enzyme digestion.
The source DNA is digested to isolate the desired gene fragment.
The vector DNA is cut to create a space for inserting the gene of interest.
This process creates fragments with compatible ends, allowing them to be joined together.

Ligation and Preparation of Recombinant DNA

To join the gene of interest with the vector DNA, the cut-out gene fragment and the cut vector with complementary ends are mixed together.
DNA ligase is added to facilitate the covalent bonding of the DNA fragments, resulting in the formation of recombinant DNA.
The ligated DNA is a combination of the vector and the gene of interest, which can be further used for transformation into host cells.

3. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction) in Recombinant DNA Technology

Polymerase Chain Reaction (PCR) is a powerful technique used to amplify specific DNA segments, including genes of interest, in vitro.
PCR allows for the rapid and precise amplification of DNA using a DNA polymerase enzyme and short, complementary DNA sequences called primers.

PCR Components

Primers: Short, chemically synthesized oligonucleotides that are complementary to specific regions of the DNA to be amplified.
DNA Polymerase: The enzyme responsible for extending the primers using nucleotides provided in the reaction and a DNA template.
Template DNA: The DNA segment that serves as the template for the synthesis of new DNA strands.
Nucleotides: The individual building blocks (A, T, C, G) that are used to extend the primers.
Buffer Solution: Provides the appropriate pH and salt conditions for the PCR reaction.

PCR Process

PCR involves a series of temperature cycles, typically consisting of three main steps:
1. Denaturation: The reaction is heated to a high temperature, causing the double-stranded DNA to separate (denature) into two single strands.
2. Annealing: The reaction is cooled to a lower temperature, allowing the primers to bind (anneal) to their complementary sequences on the single-stranded DNA.
3. Extension: The temperature is raised again, and DNA polymerase extends the primers by adding nucleotides complementary to the template strand.
These temperature cycles are repeated multiple times, resulting in the exponential amplification of the target DNA segment.
With each cycle, the number of DNA copies approximately doubles.
After a sufficient number of cycles, the desired DNA fragment is amplified to a large quantity, often on the order of billions of copies.

Thermostable DNA Polymerase

PCR relies on a special type of DNA polymerase, known as a thermostable DNA polymerase.
The most commonly used thermostable DNA polymerase is Taq polymerase, isolated from the bacterium Thermus aquaticus.
Taq polymerase remains active even during the high-temperature denaturation step, allowing PCR to be carried out through repeated cycles.

Applications

Amplified DNA fragments generated by PCR can be used for various purposes, including:

Cloning: Amplified fragments can be ligated with vectors for cloning into host cells.
Sequencing: PCR products can be sequenced to determine the nucleotide sequence of the amplified DNA.
Diagnosis: PCR is used in diagnostic tests to detect the presence of specific DNA sequences, such as disease-related genes or pathogens.
Forensics: PCR is utilized in forensic science to analyze DNA evidence from crime scenes.

4. Insertion of Recombinant DNA into the Host Cell/Organism

After creating recombinant DNA, the next crucial step is to introduce this DNA into host cells or organisms.
The method of introducing recombinant DNA into recipient cells is called transformation.
In this process, recipient cells are made competent to take up the foreign DNA, and when they do, they become transformed.

Methods of DNA Insertion

There are several methods for introducing recombinant DNA into host cells. One common method is transformation, which involves making recipient cells competent to receive and take up DNA from their surroundings.

Competency of Recipient Cells

Recipient cells must first be made competent, which means they are prepared to receive DNA.
Competency is achieved through various methods, such as treating the cells with divalent cations like calcium, which increases the efficiency of DNA uptake.
Once the cells are made competent, they can take up DNA present in their environment.

Selectable Markers

Selectable markers are genes or sequences within the recombinant DNA that confer a selective advantage to the transformed cells.
For example, if the recombinant DNA carries a gene for resistance to an antibiotic like ampicillin, the host cells become transformed into ampicillin-resistant cells.
When transformed cells are spread on agar plates containing ampicillin, only transformants (cells that have taken up the recombinant DNA) will grow, while untransformed recipient cells will die.
The ampicillin resistance gene, in this case, serves as a selectable marker because it allows for the selective growth of transformed cells in the presence of ampicillin.

Significance of Selectable Markers

Selectable markers are crucial in recombinant DNA technology as they enable the identification and selection of cells that have successfully taken up the recombinant DNA.
They provide a way to distinguish transformed cells from untransformed ones.
Selectable markers are essential for ensuring that the desired genetic modification has been introduced into the host cells.

5. Obtaining the Foreign Gene Product

In recombinant DNA technology, the ultimate goal is often to produce a desirable protein or gene product.
When a foreign DNA fragment is inserted into a cloning vector and transferred into a host cell, the foreign gene gets multiplied.
The expression of foreign genes in host cells requires specific conditions and optimization.
Large-scale production of the desired protein is often necessary for various applications.

Expression of Foreign Genes

The foreign gene introduced into the host cell is expressed under appropriate conditions, leading to the production of the target protein.
Expression of foreign genes involves technical details, including selecting suitable promoters and optimizing growth conditions.

Large-Scale Production

Large-scale production of proteins is required for various reasons, including research, industrial applications, and therapeutic purposes.
Small-scale cultures in the laboratory are insufficient for producing appreciable quantities of proteins.
To achieve high yields of the desired protein, bioreactors are used.

Bioreactors

Bioreactors are vessels designed for the large-scale production of specific products, including proteins, using microbial, plant, animal, or human cells.
Bioreactors provide optimal conditions for growth and production, including temperature, pH, substrate, salts, vitamins, and oxygen levels.
Stirred-tank bioreactors are commonly used and are equipped with various systems:
- Agitator System: Facilitates even mixing and ensures uniform distribution of nutrients and oxygen.
- Oxygen Delivery System: Ensures an adequate oxygen supply for cell growth and metabolism.
- Foam Control System: Manages foam formation, which can be a common issue during fermentation.
- Temperature Control System: Maintains the optimal temperature for cell growth and protein production.
- pH Control System: Regulates the pH level to create an environment conducive to cell growth.
- Sampling Ports: Allow for the periodic withdrawal of small culture samples for monitoring and analysis.

Stirred-Tank Reactors

Stirred-tank bioreactors are usually cylindrical or have a curved base to facilitate mixing.
They are equipped with a stirrer that promotes even mixing and oxygen availability throughout the reactor.
Alternatively, air can be bubbled through the reactor to provide oxygen.
Stirred-tank reactors are capable of producing large volumes (ranging from 100 to 1000 liters) of cultured cells and the desired protein product.

6. Downstream Processing in Recombinant DNA Technology

In recombinant DNA technology, after the biosynthetic stage where the desired gene product is produced, it goes through a series of processes before it can be marketed as a finished product.
These processes collectively form downstream processing, which involves various steps to purify, formulate, and test the product.

Key Steps in Downstream Processing

Separation and Purification: One of the primary objectives of downstream processing is to separate the desired product from other cellular components and impurities. This is typically achieved through techniques like filtration, chromatography, centrifugation, and precipitation.
Formulation: The product may need to be formulated with suitable preservatives or stabilizers to maintain its quality and stability during storage and use.
Clinical Trials: For certain products, especially in the case of drugs and biopharmaceuticals, the formulated product must undergo thorough clinical trials to evaluate its safety and efficacy in humans.
Quality Control Testing: Strict quality control testing is essential for each product to ensure it meets predefined standards for purity, potency, safety, and quality. Various analytical techniques are employed for this purpose.
Packaging: The final product is packaged in suitable containers, often under controlled conditions to prevent contamination or degradation.
Labeling and Regulatory Compliance: Proper labeling of the product is essential, including information on dosage, usage instructions, warnings, and regulatory compliance with labeling laws and regulations.
Storage and Distribution: The finished product is stored under appropriate conditions to maintain its stability until distribution to end-users.

Variability in Downstream Processing

The specific downstream processing steps and the extent of purification can vary significantly depending on the type of product being produced. For example:
- Biopharmaceuticals may require extensive purification to meet strict safety and quality standards.
- Industrial enzymes may undergo fewer purification steps.
- Research reagents may have minimal purification requirements.

Importance of Downstream Processing

Downstream processing is critical for ensuring that the final product is safe, effective, and of high quality.
It plays a crucial role in the pharmaceutical, biotechnology, and biopharmaceutical industries.
The quality and purity of the final product impact its safety, efficacy, and marketability.