Principles Of Biotechnology

Biotechnology is defined as the application of biological techniques and organisms, or parts derived from them, to produce products and processes useful to humans. Traditionally, biotechnology included processes like making curd, bread, or wine using microbes.

In a modern and more restricted sense, biotechnology refers to processes that involve using genetically modified organisms on a large scale. The European Federation of Biotechnology (EFB) provides a comprehensive definition: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Modern biotechnology is built upon two primary techniques:

1. Genetic engineering: This involves directly manipulating the genetic material (DNA or RNA) of an organism, introducing the altered genetic material into a host, and thereby changing the host's characteristics (phenotype).
2. Bioprocess engineering: This involves maintaining a sterile environment (free from unwanted microbes) in large-scale chemical engineering processes. This allows for the growth of only the desired microorganism or eukaryotic cell in large quantities for the production of valuable biotechnological products like antibiotics, vaccines, or enzymes.

Traditional breeding methods (like hybridisation in plants and animals) often lead to the transfer and multiplication of both desirable and undesirable genes. Genetic engineering techniques overcome this limitation by allowing scientists to isolate, introduce, and manipulate only specific desirable genes into a target organism without including the unwanted ones.

When a piece of foreign DNA is introduced into an alien organism, it usually cannot multiply on its own. However, if this foreign DNA is integrated into the host organism's genome, it can replicate and be inherited along with the host DNA. This is because the foreign DNA becomes part of a chromosome, which has the ability to replicate.

Chromosomes contain specific DNA sequences called the origin of replication (ori), which are essential for initiating DNA replication. For any foreign DNA piece to replicate and multiply within a host organism, it must be linked to an origin of replication. Linking foreign DNA to an origin of replication allows it to be replicated and multiplied in the host, a process also referred to as cloning (making multiple identical copies of the DNA).

The first artificial recombinant DNA molecule was constructed in 1972 by Stanley Cohen and Herbert Boyer. They achieved this by linking a gene encoding antibiotic resistance to a plasmid (a small, circular, extra-chromosomal DNA that replicates autonomously) from the bacterium Salmonella typhimurium.

Steps involved in their construction:

1. They isolated the antibiotic resistance gene by precisely cutting a piece of DNA from a plasmid using 'molecular scissors' called restriction enzymes.
2. They cut the plasmid DNA (vector) using the same restriction enzyme.
3. The isolated antibiotic resistance gene was then joined (ligated) with the cut plasmid DNA using the enzyme DNA ligase. This created a novel combination of circular DNA, called recombinant DNA, which could replicate autonomously.
4. This recombinant DNA was then transferred into Escherichia coli (a related bacterium).

Once inside the host *E. coli*, the recombinant DNA replicated using the host cell's machinery, making multiple copies of the antibiotic resistance gene. This process of multiplying the antibiotic resistance gene in *E. coli* is termed cloning of the antibiotic resistance gene.

From this, the basic steps involved in genetically modifying an organism using recombinant DNA technology can be outlined:

1. Identifying and isolating DNA containing the desired gene(s).
2. Introducing the isolated desired DNA into a suitable host organism.
3. Ensuring the introduced DNA is maintained in the host (replicates) and is transferred to its progeny.

Tools Of Recombinant Dna Technology

Recombinant DNA technology relies on several key molecular tools:

1. Restriction enzymes
2. Polymerase enzymes
3. DNA ligases
4. Vectors
5. Host organism

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Restriction Enzymes

Restriction enzymes were first isolated in 1963 from *Escherichia coli*. They were found to be responsible for restricting the growth of bacteriophages (viruses that infect bacteria) by cutting the viral DNA. One enzyme added methyl groups to DNA, while the other cut the DNA. The cutting enzyme was named restriction endonuclease.

The first restriction endonuclease whose function was dependent on a specific DNA sequence, Hind II, was isolated and characterised five years later. Hind II always cut DNA at a specific sequence of six base pairs, known as its recognition sequence.

Hundreds of restriction enzymes have since been isolated from different bacteria, each recognising a unique DNA sequence. Restriction enzymes belong to a larger class of enzymes called nucleases, which break phosphodiester bonds in nucleic acid chains. Nucleases are of two types:

• Exonucleases: Remove nucleotides one by one from the ends of a DNA molecule.
• Endonucleases: Make cuts at specific positions within a DNA molecule.

Each restriction endonuclease functions by scanning the DNA sequence until it finds its specific recognition site. It then binds to the DNA and cleaves the sugar-phosphate backbone of both strands at specific points within or near the recognition site.

Many restriction enzyme recognition sites are palindromic nucleotide sequences. A DNA palindrome reads the same forwards on one strand ($5' \to 3'$) as it does backwards on the complementary strand ($3' \to 5'$), when read with the same orientation ($5' \to 3'$).

Restriction enzymes typically cut the DNA strands slightly away from the center of the palindrome, but between the same two bases on opposite strands. This staggered cut results in single-stranded overhangs at the ends of the DNA fragments. These overhangs are called sticky ends because they can form hydrogen bonds with complementary sticky ends of other DNA fragments cut by the same enzyme.

Sticky ends are advantageous in recombinant DNA technology because they facilitate the joining (ligation) of DNA fragments from different sources using DNA ligase. When a vector and a foreign DNA fragment are cut with the same restriction enzyme, they generate complementary sticky ends, allowing them to be ligated together to form a recombinant DNA molecule.

Separation and isolation of DNA fragments: After digestion by restriction enzymes, the resulting DNA fragments can be separated based on their size using a technique called gel electrophoresis. Since DNA fragments are negatively charged due to the phosphate groups, they migrate towards the positive electrode (anode) when placed in an electric field.

The separation occurs within a matrix, most commonly an agarose gel (a natural polymer from seaweeds). Smaller DNA fragments move faster and farther through the gel matrix than larger fragments, due to a sieving effect.

DNA fragments are invisible in visible light. They are visualised by staining the gel with a fluorescent dye like ethidium bromide and exposing it to UV radiation. DNA bands appear as bright orange under UV light.

The desired DNA fragment bands are cut out from the gel (a process called elution) and the DNA is extracted from the gel piece. These purified DNA fragments are then used for constructing recombinant DNA by ligating them into cloning vectors.

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Cloning Vectors

A cloning vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. Plasmids and bacteriophages are commonly used as vectors because they can replicate autonomously within bacterial cells, independent of the host chromosomal DNA.

Bacteriophages often have very high copy numbers (many copies per cell), and some plasmids also have high copy numbers (15-100 copies per cell, sometimes higher). By inserting a foreign piece of DNA into such a vector, the foreign DNA can be amplified to the same copy number as the vector within the host cell.

Modern vectors are engineered with features to facilitate easy insertion of foreign DNA and selection of host cells that have taken up the recombinant DNA (transformants).

Essential features of a cloning vector:

1. Origin of replication (ori): This is a specific DNA sequence where replication begins. Any DNA sequence linked to the 'ori' sequence can initiate replication within the host cell. The 'ori' sequence also controls the copy number of the linked DNA; vectors with 'ori' that support high copy number are used to obtain many copies of the desired DNA.
2. Selectable marker: A gene within the vector that allows for the identification and selection of host cells that have successfully taken up the vector (transformed cells) from those that have not (non-transformants). Selectable markers provide a trait to the transformant that is not present in the recipient cell. Genes conferring resistance to antibiotics (like ampicillin, chloramphenicol, tetracycline, kanamycin) are commonly used selectable markers in *E. coli*. Normal *E. coli* cells are susceptible to these antibiotics, but transformants carrying the vector with the resistance gene can grow in the presence of the antibiotic.
3. Cloning sites (Recognition sites): The vector should ideally have very few, preferably unique (single), recognition sites for commonly used restriction enzymes. Multiple recognition sites for the same enzyme within the vector would lead to fragmentation of the vector itself, complicating the cloning process. Ligation of foreign DNA is usually performed at a restriction site located within a selectable marker gene or a gene encoding a protein (like an enzyme).
   • Example: Inserting foreign DNA at the *BamH* I site within the tetracycline resistance gene (tetR) of the vector pBR322. If foreign DNA is successfully ligated here, the tetR gene is inactivated (insertional inactivation). Host cells transformed with this recombinant vector will be resistant to ampicillin (ampR gene is intact) but lose resistance to tetracycline. To select recombinants, transformed cells are first plated on a medium with ampicillin (to select for transformants), and then colonies are replica plated onto a medium containing tetracycline. Cells with recombinant plasmids grow on ampicillin but not on tetracycline, while non-recombinant transformants (with intact tetR gene) grow on both.
   An alternative to this cumbersome method is using insertional inactivation in a gene that produces a detectable product, like an enzyme. For example, inserting foreign DNA into a gene encoding β-galactosidase in the vector. In the presence of a chromogenic substrate, bacteria with a non-recombinant plasmid (intact β-galactosidase gene) will produce a blue colour. Bacteria with a recombinant plasmid (inactivated β-galactosidase gene due to insertional inactivation) will not produce colour and appear white. This allows for easy differentiation of recombinants.

Vectors for cloning genes in plants and animals: Nature has provided examples of how to transfer genes into eukaryotic cells. Pathogens like *Agrobacterium tumifaciens* and retroviruses have evolved mechanisms to deliver their genetic material into host cells and alter their functions.

• *Agrobacterium tumifaciens*, a bacterium causing tumors in dicot plants, transfers a piece of its DNA, called T-DNA, from its Tumor inducing (Ti) plasmid into the plant genome, inducing tumor formation and directing the plant cells to produce substances needed by the bacterium. The Ti plasmid has been modified into a disarmed cloning vector that is no longer pathogenic but retains the ability to deliver genes of interest into various plants.
• Retroviruses, which can transform normal animal cells into cancerous cells, have also been 'disarmed' (made non-pathogenic) and used as vectors to deliver desirable genes into animal cells (e.g., in gene therapy).

Once a gene of interest is ligated into a suitable vector, the recombinant DNA is transferred into a host organism (bacterium, plant, or animal) for replication and expression.

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Competent Host (For Transformation With Recombinant Dna)

DNA is a hydrophilic molecule and cannot easily cross the hydrophobic cell membrane. Therefore, the host cell must be made 'competent' to take up the recombinant DNA.

Methods to make bacterial cells competent:

• Treatment with a specific concentration of a divalent cation (e.g., calcium ions). This increases the permeability of the cell membrane and wall to DNA.
• Incubating the cells with recombinant DNA on ice, followed by a brief exposure to $42^\circ\textsf{C}$ (heat shock), and then returning them to ice. This heat shock treatment facilitates the uptake of DNA by the bacteria.

Other methods for introducing alien DNA into host cells (especially for plant and animal cells):

• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell using a fine needle.
• Biolistics (Gene gun): Suitable for plant cells. Cells are bombarded with high-velocity micro-particles (usually gold or tungsten) coated with DNA.
• Using disarmed pathogen vectors: Non-pathogenic versions of viruses or bacteria that naturally infect cells can be used to deliver the recombinant DNA into the host cells (as mentioned with *Agrobacterium* and retroviruses).

Processes Of Recombinant Dna Technology

Recombinant DNA technology involves several distinct steps performed in a specific order:

1. Isolation of the genetic material (DNA).
2. Fragmentation of DNA using restriction endonucleases.
3. Isolation of the desired DNA fragment (gene of interest).
4. Ligation of the DNA fragment into a suitable vector.
5. Transferring the recombinant DNA into a host cell/organism (Transformation).
6. Culturing the host cells on a large scale to obtain the desired product.
7. Extraction and purification of the desired product.

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Isolation Of The Genetic Material (Dna)

DNA is the genetic material in most organisms. To work with DNA (e.g., cut it with restriction enzymes), it must be isolated in a pure form, free from other cellular components.

Steps for DNA isolation:

• The cell wall and cell membrane must be broken open to release the DNA and other macromolecules (RNA, proteins, polysaccharides, lipids). This is achieved by treating the cells/tissues with specific enzymes:
  • Lysozyme: Used for bacterial cells.
  • Cellulase: Used for plant cells.
  • Chitinase: Used for fungal cells.
• RNA is removed by treatment with the enzyme Ribonuclease (RNase).
• Proteins are removed by treatment with the enzyme Protease. Histones, which are tightly associated with DNA, are also digested.
• Other molecules are removed by appropriate treatments.
• Finally, purified DNA is precipitated out of the solution by adding chilled ethanol. DNA is insoluble in cold ethanol and appears as fine threads that can be collected by 'spooling' (winding onto a rod).

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Cutting Of Dna At Specific Locations

Once purified, the DNA (both the source DNA containing the gene of interest and the vector DNA) is subjected to digestion with specific restriction enzymes. The digestion is performed by incubating the DNA with the enzyme under optimal conditions (temperature, pH, buffer). The progress of the digestion can be monitored using agarose gel electrophoresis.

After digestion, the desired DNA fragment (gene of interest) is isolated from the source DNA using gel electrophoresis and elution, as described earlier.

The isolated gene of interest (insert DNA) is then joined with the cut vector DNA. This joining process, called ligation, is catalysed by the enzyme DNA ligase. DNA ligase forms phosphodiester bonds between the sugar-phosphate backbones of the cut DNA fragments, creating a continuous recombinant DNA molecule.

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Amplification Of Gene Of Interest Using Pcr

Often, the amount of isolated DNA containing the gene of interest is very small. To obtain sufficient quantities for ligation and cloning, the gene can be amplified *in vitro* using the Polymerase Chain Reaction (PCR).

PCR is a technique for making multiple copies of a specific DNA sequence. It involves using:

• Two sets of primers: Short, chemically synthesised oligonucleotide sequences complementary to the regions flanking the target DNA sequence.
• A thermostable DNA polymerase: An enzyme (like Taq polymerase isolated from *Thermus aquaticus*) that can withstand the high temperatures required for DNA denaturation during the process.
• Deoxyribonucleotides (dATP, dGTP, dCTP, dTTP) as building blocks.

The PCR process consists of repeated cycles (typically 20-35 cycles), each involving three main steps:

1. Denaturation: The double-stranded target DNA is heated to a high temperature ($\sim 94-98^\circ\textsf{C}$) to separate the two strands.
2. Annealing: The temperature is lowered ($\sim 50-65^\circ\textsf{C}$) to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates.
3. Extension (Polymerisation): The temperature is raised to the optimal temperature for the thermostable DNA polymerase ($\sim 72^\circ\textsf{C}$). The polymerase extends the primers, synthesising new complementary DNA strands.

Each PCR cycle doubles the number of DNA copies. After 'n' cycles, the target DNA sequence is amplified $2^n$ times. Thus, PCR can amplify a small amount of DNA to billions of copies (approximately a billion times amplification after 30 cycles, $2^{30} \approx 10^9$). The amplified DNA fragment can then be ligated into a vector for cloning.

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Insertion Of Recombinant Dna Into The Host Cell/Organism

Once the recombinant DNA molecule is constructed, it needs to be introduced into a suitable host cell. This process is called transformation (for bacteria) or transfection (for eukaryotic cells).

Methods to facilitate the uptake of recombinant DNA by host cells (after making them competent) were described earlier: chemical treatment with divalent cations and heat shock, micro-injection, biolistics (gene gun), and using disarmed pathogen vectors.

A recombinant DNA carrying a selectable marker gene (e.g., antibiotic resistance) allows for the selection of transformed cells. If transformed *E. coli* cells (carrying the ampicillin resistance gene) are plated on a medium containing ampicillin, only those cells that have taken up the recombinant DNA will survive and grow, while the untransformed cells will die. The selectable marker allows for the identification and isolation of successful transformants.

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Obtaining The Foreign Gene Product

The ultimate goal of most recombinant DNA technologies is to express the foreign gene inserted into the host cell and obtain the desired product, typically a protein.

When a foreign gene is expressed in a host cell that normally does not contain that gene, the resulting protein is called a recombinant protein.

Host cells containing the cloned gene are grown under conditions that optimise the expression of the target protein. Initially, this may be done on a small scale in the laboratory.

To produce large quantities of the desired protein, the host cells are cultured on a large scale, typically in vessels called bioreactors. Bioreactors can handle large volumes of culture (100-1000 litres or more).

Bioreactors provide controlled optimal conditions for cell growth and product formation, including temperature, pH, substrate concentration, salt concentration, vitamin supply, and oxygen availability.

Stirred-tank bioreactors are commonly used. They are designed (cylindrical or with a curved base) to ensure even mixing of the culture contents. A stirrer (agitator) mixes the medium and cells and also facilitates oxygen transfer throughout the reactor. Sterile air can be bubbled through the reactor (sparged stirred-tank bioreactor) to enhance oxygen availability.

Bioreactors often have systems for monitoring and controlling parameters like temperature, pH, foam, and oxygen levels. Sampling ports allow for periodic withdrawal of small culture volumes to check the progress.

Continuous culture systems can be used in bioreactors, where fresh medium is continuously added while used medium is drained. This keeps the cells in their active exponential growth phase, leading to larger biomass and higher product yields compared to batch cultures.

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Downstream Processing

After the desired product (e.g., recombinant protein) is produced in the bioreactor (biosynthetic stage), it needs to be separated, purified, and formulated for market use. These steps are collectively known as downstream processing.

Downstream processing involves:

1. Separation: Separating the product from the cells and the culture medium.
2. Purification: Removing contaminants and isolating the pure product. This may involve various chromatography techniques, filtration, etc.
3. Formulation: Preparing the purified product into a suitable formulation, often adding preservatives.

For products intended for medical use (like drugs), formulation must undergo thorough clinical trials to ensure safety and efficacy.

Strict quality control testing is mandatory for each product to ensure purity, safety, and consistency. The specific steps and procedures in downstream processing and quality control vary depending on the nature of the product.

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