Chapter Notes

Biotechnology : Principles and Processes

Biotechnology uses live organisms, or enzymes from them, to create products and processes that are useful to humans. While traditional processes like making curd, bread, or wine are technically forms of biotechnology, the modern definition focuses on using genetically modified organisms (GMOs) to achieve these results on a much larger 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'.

Principles of Biotechnology

Modern biotechnology is built on two core techniques:

1. Genetic engineering: This involves altering the chemistry of genetic material (DNA and RNA) and introducing it into a host organism. The goal is to change the phenotype (observable characteristics) of the host.
2. Bioprocess engineering: This involves maintaining a sterile, contamination-free environment in large-scale manufacturing processes. This ensures that only the desired cells (microbial, eukaryotic) grow, allowing for the production of biotechnological products like antibiotics, vaccines, and enzymes.

Conceptual Development of Genetic Engineering

Traditional plant and animal breeding often leads to the transfer of undesirable genes along with the desired ones. Genetic engineering overcomes this limitation. It allows us to isolate and introduce only one or a specific set of desirable genes into a target organism. This is achieved through techniques like creating recombinant DNA, gene cloning, and gene transfer.

For an alien piece of DNA to multiply within a host organism, it must become part of the host's chromosome or have its own origin of replication (ori). The ori is a specific DNA sequence that initiates replication. By linking a foreign DNA segment to an ori, we can make it replicate and create multiple identical copies within the host. This process is called cloning.

The first artificial recombinant DNA (rDNA) molecule was created in 1972 by Stanley Cohen and Herbert Boyer.

1. They isolated an antibiotic resistance gene from a plasmid (a small, circular, self-replicating DNA molecule) of Salmonella typhimurium.
2. They used restriction enzymes, also known as 'molecular scissors', to cut the DNA at specific locations.
3. The cut piece of DNA (the gene) was then linked to a plasmid vector using an enzyme called DNA ligase. A vector is a DNA molecule used to carry foreign genetic material into another cell.
4. This newly created rDNA was transferred into Escherichia coli (E. coli), a bacterium where it could replicate and make multiple copies of the antibiotic resistance gene.

This breakthrough laid the foundation for modern biotechnology.

There are three basic steps to genetically modifying an organism:

1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host.
3. Maintenance of the introduced DNA in the host and its transfer to the next generation (progeny).

Tools of Recombinant DNA Technology

To perform genetic engineering, we need a set of key tools:

• Restriction enzymes
• Polymerase enzymes
• Ligases
• Vectors
• A host organism

Restriction Enzymes

These enzymes are like 'molecular scissors' that cut DNA. In 1963, two enzymes were isolated from E. coli that restricted the growth of bacteriophages (viruses that infect bacteria). One added methyl groups to DNA, and the other cut it. The one that cut DNA was named a restriction endonuclease.

The first restriction endonuclease to be fully characterized was Hind II. It was found to always cut DNA at a specific sequence of six base pairs, known as its recognition sequence. Today, over 900 restriction enzymes have been isolated from more than 230 strains of bacteria.

Naming Convention:

• The first letter comes from the genus of the organism (e.g., E from Escherichia).
• The next two letters come from the species (e.g., co from coli).
• The next letter represents the strain (e.g., R from RY 13).
• A Roman numeral indicates the order of discovery (e.g., I for the first one found).
• Example: EcoRI comes from Escherichia coli RY 13.

Restriction enzymes belong to a larger class called nucleases.

• Exonucleases remove nucleotides from the ends of the DNA.
• Endonucleases make cuts at specific positions within the DNA.

Each restriction endonuclease recognizes a specific palindromic nucleotide sequence. A palindrome in DNA is a sequence of base pairs that reads the same on both strands when read in the same orientation (e.g., $5^{\prime} \rightarrow 3^{\prime}$).

For example, the recognition site for EcoRI is:

The enzyme cuts the DNA strand a little away from the center of the palindrome, but between the same two bases on opposite strands. This creates overhanging single-stranded portions called sticky ends. These ends are 'sticky' because they can form hydrogen bonds with their complementary counterparts. This stickiness helps the enzyme DNA ligase to join DNA fragments.

Separation and Isolation of DNA Fragments

After cutting DNA with restriction enzymes, the resulting fragments need to be separated. This is done using a technique called gel electrophoresis.

1. Principle: DNA fragments are negatively charged. When an electric field is applied, they move towards the positive electrode (anode).
2. Matrix: The fragments move through a gel matrix, most commonly agarose (a natural polymer from sea weeds).
3. Separation: The agarose gel acts like a sieve. Smaller DNA fragments move farther and faster through the gel than larger fragments.
4. Visualization: Pure DNA is not visible to the naked eye. The fragments are stained with a compound called ethidium bromide and then exposed to UV radiation. This makes the DNA appear as bright orange-colored bands.
5. Elution: The desired band of DNA is cut out from the gel, and the DNA is extracted from the gel piece. This process is called elution. The purified DNA fragment is now ready to be used to construct recombinant DNA.

Cloning Vectors

Vectors are DNA molecules that can carry a foreign DNA segment and replicate inside a host cell. Plasmids and bacteriophages are commonly used vectors because they can replicate independently of the host's chromosomal DNA. They can have a high copy number (from 1-2 to 15-100 or more copies per cell), which allows for the amplification of the linked foreign DNA.

To be a useful vector, it must have the following features:

• Origin of replication (ori): This is the DNA sequence where replication begins. Any piece of DNA linked to this sequence will be replicated within the host. The ori also controls the copy number of the linked DNA. To get many copies of a target gene, it should be cloned in a vector with an ori that supports a high copy number.

• Selectable marker: This helps in identifying and selecting cells that have been successfully transformed (i.e., have taken up the vector) and eliminating non-transformants. Genes that provide resistance to antibiotics like ampicillin, tetracycline, or kanamycin are common selectable markers for E. coli.

• Cloning sites: These are specific recognition sites where restriction enzymes can cut the vector to insert the foreign DNA. A good vector should have very few, preferably single, recognition sites for commonly used restriction enzymes. Having multiple sites would complicate the process by generating several fragments.

Insertional Inactivation This is a method to screen for recombinant plasmids.

• Example using pBR322: The vector pBR322 has two antibiotic resistance genes: one for ampicillin ($amp^R$) and one for tetracycline ($tet^R$).
• If we insert our foreign DNA into a restriction site located within the $tet^R$ gene (e.g., the BamH I site), the gene becomes inactivated.
• The resulting recombinant plasmid will still have the $amp^R$ gene but will lose its resistance to tetracycline.
• Screening:
  1. Cells are grown on a medium with ampicillin. Only transformed cells (both recombinant and non-recombinant) will survive.
  2. These colonies are then transferred to a medium with tetracycline.
  3. Non-recombinant cells will grow (they have both resistances), but recombinant cells will die (they lost tetracycline resistance).
  4. By comparing the two plates, the recombinant colonies can be identified.

An alternative method uses a marker that produces a color. For example, if a foreign gene is inserted into the coding sequence of the enzyme $\beta$-galactosidase, the enzyme is inactivated. When grown on a special medium, non-recombinant colonies will appear blue, while recombinant colonies will be white. This is called blue-white screening.

Vectors for Cloning Genes in Plants and Animals

Nature has already provided us with tools for gene transfer.

• For Plants: The bacterium Agrobacterium tumifaciens is a natural genetic engineer. It contains the Ti plasmid (tumor-inducing), which can transfer a piece of its DNA (called 'T-DNA') into plant cells, causing tumors. Scientists have "disarmed" this plasmid by removing its tumor-causing genes, turning it into an efficient vector for delivering genes of interest into many plant species.
• For Animals: Retroviruses can transform normal animal cells into cancerous ones. These viruses have also been "disarmed" and are now used as vectors to deliver desirable genes into animal cells.

Competent Host (For Transformation with Recombinant DNA)

DNA is a hydrophilic (water-loving) molecule, so it cannot easily pass through the cell membrane, which is made of lipids. Therefore, bacterial cells must be made competent to take up foreign DNA.

• Heat Shock Method:
  1. Cells are treated with a specific concentration of a divalent cation, like calcium ($Ca^{2+}$), which increases the permeability of the cell wall.
  2. The cells are incubated with the recombinant DNA on ice.
  3. They are then briefly placed at $42^{\circ}\text{C}$ (heat shock).
  4. Finally, they are put back on ice. This process forces the bacteria to take up the recombinant DNA.

Other methods for introducing foreign DNA include:

• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics or Gene Gun: Tiny micro-particles of gold or tungsten coated with DNA are shot at high velocity into plant cells.
• Disarmed Pathogen Vectors: Modified viruses or bacteria are used to "infect" the host cell and transfer the recombinant DNA.

Processes of Recombinant DNA Technology

Recombinant DNA technology involves a sequence of steps:

1. Isolation of the Genetic Material (DNA): The DNA must be obtained in a pure form.

   • The cell wall is broken down using enzymes: lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi.
   • RNA is removed by treating with ribonuclease.
   • Proteins (like histones) are removed by treating with protease.
   • After other molecules are removed, purified DNA is precipitated by adding chilled ethanol. The DNA appears as a collection of fine threads in the suspension, which can be spooled out.

2. Cutting of DNA at Specific Locations: The purified DNA and the vector DNA are incubated with the same restriction enzyme under optimal conditions. Gel electrophoresis can be used to check that the DNA has been successfully cut.

3. Ligation of DNA Fragment into a Vector: The 'gene of interest' and the cut vector are mixed together, and the enzyme DNA ligase is added. This enzyme joins the sticky ends, creating a recombinant DNA molecule.

4. Amplification of Gene of Interest using PCR: PCR stands for Polymerase Chain Reaction. It is a technique to synthesize multiple copies of a specific gene or DNA segment in vitro (in a test tube).

   • Requirements:
     • Primers: Two sets of small, chemically synthesized DNA sequences that are complementary to the regions flanking the target DNA.
     • DNA polymerase: A special thermostable enzyme (like Taq polymerase from the bacterium Thermus aquaticus) that can withstand high temperatures.
     • Nucleotides.
   • The PCR Cycle:
     1. Denaturation: The double-stranded DNA is heated to a high temperature to separate the two strands.
     2. Annealing: The temperature is lowered to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA.
     3. Extension: The DNA polymerase extends the primers, using the nucleotides to synthesize a new complementary strand.
   • This cycle is repeated about 30 times, leading to an exponential amplification of the target DNA, producing about a billion copies.

5. Insertion of Recombinant DNA into the Host Cell/Organism: The ligated DNA is introduced into a competent host cell (e.g., E. coli). This process is called transformation. If the recombinant DNA contains a selectable marker (like an ampicillin resistance gene), only the transformed cells will grow on a medium containing that antibiotic.

6. Obtaining the Foreign Gene Product: The ultimate aim of recombinant technology is often to produce a desirable protein, called a recombinant protein.

   • Culturing: The cells containing the cloned gene are grown. In a continuous culture system, used medium is drained out while fresh medium is added, keeping the cells in their most active growth phase (log/exponential phase) to produce a large biomass.
   • Bioreactors: For large-scale production (100-1000 litres), bioreactors are used. These are large vessels that provide optimal growth conditions (temperature, pH, substrate, oxygen, etc.). The most common type is the stirred-tank bioreactor, which has a stirrer for even mixing and oxygen distribution, an oxygen delivery system, and controls for temperature, pH, and foam.

7. Downstream Processing: After the product is synthesized, it must be prepared for marketing. This final stage is called downstream processing.

   • It includes separation and purification of the product.
   • The product is then formulated with suitable preservatives.
   • If the product is a drug, it must undergo rigorous clinical trials.
   • Strict quality control testing is required for every batch. The specific processes vary depending on the product.