Biotechnology : Principles and Processes

Understanding Biotechnology

Biotechnology, a field that emerged from modern biology in the twentieth century, has significantly improved health and food production. In a broad sense, it includes microbe-mediated processes like making curd, bread, or wine. However, it is primarily used today to refer to processes using genetically modified organisms on a larger scale. It also encompasses techniques like in vitro fertilisation, gene synthesis, DNA vaccine development, and gene correction.

The European Federation of Biotechnology (EFB) defines biotechnology as: ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

Historically, human knowledge, especially in natural sciences, has been anthropocentric, aiming to develop technologies for human comfort and welfare, with biology’s main utility being a source of food.

Herbert Boyer: A Pioneer in Biotechnology

Herbert Boyer (born 1936) is a pivotal figure in biotechnology.

• He completed his graduate work at the University of Pittsburgh in 1963 and post-graduate studies at Yale.
• In 1966, he became an assistant professor at the University of California at San Francisco.
• By 1969, Boyer studied restriction enzymes from E. coli bacteria, observing their ability to cut DNA strands in a specific manner, leaving ‘sticky ends’. These sticky ends allowed precise pasting of DNA pieces together.
• This discovery led to a collaboration with Stanley Cohen, who had developed a method to remove and reinsert small DNA ringlets called plasmids from bacterial cells.
• By combining DNA splicing with Cohen’s plasmid reinsertion technique, Boyer and Cohen were able to recombine DNA segments in desired configurations and insert them into bacterial cells, turning them into “manufacturing plants for specific proteins”.
• This breakthrough laid the foundation for the discipline of biotechnology.

Principles of Biotechnology

Modern biotechnology is underpinned by two core techniques:

1. Genetic Engineering: Involves techniques to alter the chemistry of genetic material (DNA and RNA), introduce these into host organisms, and thereby change the host organism’s phenotype.
2. Bioprocess Engineering: Focuses on maintaining a sterile (microbial contamination-free) environment in chemical engineering processes. This ensures the growth of only the desired microbe or eukaryotic cell in large quantities for manufacturing biotechnological products like antibiotics, vaccines, and enzymes.

Conceptual Development of Genetic Engineering

Genetic engineering addresses limitations of traditional hybridisation procedures, which often lead to the inclusion of undesirable genes alongside desired ones.

• Sexual reproduction offers variations and unique genetic combinations, some beneficial, while asexual reproduction preserves genetic information.
• Genetic engineering techniques, including recombinant DNA creation, gene cloning, and gene transfer, overcome this by allowing isolation and introduction of only specific desirable genes into a target organism.

The Fate of Alien DNA

When a piece of alien DNA is transferred into an organism, it typically cannot multiply itself unless it integrates into the recipient’s genome. If it becomes part of a chromosome, it can multiply and be inherited with the host DNA.

• A chromosome contains a specific DNA sequence called the origin of replication (ori), which initiates replication.
• For alien DNA to multiply in a host, it must be linked with an origin of replication. This process is known as cloning, creating multiple identical copies of a template DNA.

Construction of the First Artificial Recombinant DNA

In 1972, Stanley Cohen and Herbert Boyer successfully constructed the first artificial recombinant DNA molecule.

• They linked a gene encoding antibiotic resistance with a native plasmid (an autonomously replicating circular extra-chromosomal DNA) from Salmonella typhimurium.
• They isolated the antibiotic resistance gene by cutting a piece of DNA from a plasmid using ‘molecular scissors’ – restriction enzymes.
• The cut DNA piece was then linked with the plasmid DNA using the enzyme DNA ligase, which joins cut DNA ends. These plasmids act as vectors to transfer the DNA piece.
• This resulted in a new combination of circular, autonomously replicating DNA created in vitro, known as recombinant DNA.
• When transferred into Escherichia coli, this recombinant DNA replicated using the host’s DNA polymerase, leading to the cloning of the antibiotic resistance gene.

Basic Steps in Genetically Modifying an Organism

There are three fundamental steps in genetically modifying an organism:

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

Tools of Recombinant DNA Technology

Genetic engineering, or recombinant DNA technology, relies on several key tools: restriction enzymes, polymerase enzymes, ligases, vectors, and the host organism.

Restriction Enzymes

In 1963, two enzymes restricting bacteriophage growth in Escherichia coli were isolated: one added methyl groups to DNA, and the other, called restriction endonuclease, cut DNA.

• The first restriction endonuclease, Hind II, isolated five years later, was found to consistently cut DNA at a specific six base pair sequence, known as its recognition sequence.
• Currently, over 900 restriction enzymes have been isolated from more than 230 bacterial strains, each recognising different sequences.

Naming Convention for Restriction Enzymes:

• The first letter is from the genus (e.g., ‘E’ from Escherichia).
• The next two letters are from the species (e.g., ‘co’ from coli).
• A letter (like ‘R’ in EcoRI) may indicate the strain (e.g., RY 13).
• Roman numerals denote the order of isolation from that strain (e.g., ‘I’ in EcoRI).

Types of Nucleases: Restriction enzymes belong to a larger class called nucleases, which are of two kinds:

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

How Restriction Endonucleases Function:

• Each restriction endonuclease “inspects” the DNA length for its specific palindromic nucleotide sequence.
• A DNA palindrome is a sequence of base pairs that reads the same on both strands when read in the same orientation (e.g., 5’-GAATTC-3’ and 3’-CTTAAG-5’).
• The enzyme binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.
• Cuts are made a little away from the centre of the palindrome sites but between the same two bases on opposite strands, leaving single-stranded overhangs called sticky ends.
• These sticky ends can form hydrogen bonds with complementary cut counterparts, facilitating the action of DNA ligase.
• Restriction endonucleases are used in genetic engineering to form recombinant DNA molecules by joining DNA from different sources, especially when cut by the same enzyme, as they produce identical sticky ends.

Separation and Isolation of DNA Fragments

After DNA is cut by restriction endonucleases into fragments, these fragments can be separated using gel electrophoresis.

• DNA fragments are negatively charged, so they migrate towards the positive electrode (anode) under an electric field.
• The most common matrix used is agarose gel, a natural polymer from seaweeds.
• Fragments separate based on size due to a sieving effect: smaller fragments move farther.
• Separated DNA fragments are not visible in visible light; they are visualised by staining with ethidium bromide and exposure to UV radiation, appearing as bright orange bands.
• The separated bands are then cut from the gel and extracted in a step called elution.
• The purified DNA fragments are then used to construct recombinant DNA by joining them with cloning vectors.

Cloning Vectors

Plasmids and bacteriophages are commonly used as cloning vectors because they can replicate independently within bacterial cells.

• Bacteriophages typically have very high copy numbers of their genome per cell.
• Plasmids can have varying copy numbers, from one or two up to 15-100 or even higher per cell.
• Linking an alien DNA piece with a bacteriophage or plasmid DNA allows its multiplication equal to the vector’s copy number.
• Modern vectors are engineered for easy linking of foreign DNA and efficient selection of recombinants (cells containing the desired recombinant DNA) from non-recombinants (cells without the recombinant DNA).

Features Required to Facilitate Cloning into a Vector:

1. Origin of replication (ori):

   • This is the sequence where DNA replication starts.
   • Any DNA linked to this sequence can replicate within the host cell.
   • The ori sequence also controls the copy number of the linked DNA; to recover many copies of target DNA, it should be cloned in a vector with a high copy number ori.

2. Selectable Marker:

   • Helps identify and eliminate non-transformants (cells that haven’t taken up DNA) and selectively permit the growth of transformants (cells that have taken up DNA).
   • Genes encoding resistance to antibiotics (e.g., ampicillin, chloramphenicol, tetracycline, kanamycin) are useful selectable markers for E. coli, as normal E. coli cells do not have resistance to these antibiotics.

3. Cloning Sites:

   • The vector needs very few, preferably single, recognition sites for commonly used restriction enzymes.
   • Multiple recognition sites within the vector would generate several fragments, complicating gene cloning.
   • Ligation of alien DNA is often carried out at a restriction site located within one of the antibiotic resistance genes. For example, in the vector pBR322 (shown in Figure 9.4 in the source), foreign DNA can be ligated at the BamH I site within the tetracycline resistance gene.
   • Selection using antibiotic resistance inactivation: If foreign DNA is inserted into an antibiotic resistance gene (e.g., tetracycline resistance), the recombinant plasmid will lose that resistance.
     • Transformants are first plated on a medium containing one antibiotic (e.g., ampicillin, for which the transformants still have resistance).
     • Then, they are transferred to a medium containing the second antibiotic (e.g., tetracycline).
     • Recombinants will grow on the first medium but not the second.
     • Non-recombinants (which did not incorporate the foreign DNA and thus retain both resistances) will grow on both media.
     • This method is cumbersome as it requires simultaneous plating on two different antibiotic media.
   • Alternative selectable markers (insertional inactivation with chromogenic substrate):
     • These differentiate recombinants based on their ability to produce colour.
     • Recombinant DNA is inserted into the coding sequence of an enzyme, such as β-galactosidase, leading to insertional inactivation of the enzyme’s gene.
     • If the plasmid in the bacteria does not have an insert, the β-galactosidase gene is active, and in the presence of a chromogenic substrate, the colonies will be blue-coloured.
     • If an insert is present, the β-galactosidase gene is inactivated, and the colonies will not produce any colour, thus identifying them as recombinant colonies.

4. Vectors for Cloning Genes in Plants and Animals:

   • Nature has provided examples of gene transfer: bacteria and viruses have long known how to deliver genes to eukaryotic cells and manipulate them.
   • Agrobacterium tumifaciens: A pathogen of dicot plants, it delivers a piece of DNA called ‘T-DNA’ to transform normal plant cells into tumors and direct them to produce chemicals needed by the pathogen.
   • Retroviruses: In animals, they can transform normal cells into cancerous cells.
   • Scientists have leveraged this knowledge to transform these pathogen tools into useful vectors.
     • The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has been modified to be non-pathogenic but still capable of delivering genes of interest into plants.
     • Similarly, retroviruses have been disarmed and are now used to deliver desirable genes into animal cells.
   • Once a gene is ligated into a suitable vector, it is transferred into a bacterial, plant, or animal host for multiplication.

Competent Host (For Transformation with Recombinant DNA)

DNA is a hydrophilic molecule and cannot easily pass through cell membranes. To force bacteria to take up plasmids, they must be made ‘competent’.

• This is achieved by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency of DNA entry through pores in the cell wall.
• Recombinant DNA is then forced into these cells by incubating them on ice, briefly subjecting them to heat shock (42°C), and then returning them to ice. This allows the bacteria to take up the DNA.

Other Methods to Introduce Alien DNA into Host Cells:

• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics or Gene Gun: For plants, cells are bombarded with high-velocity micro-particles (gold or tungsten) coated with DNA.
• Disarmed Pathogen Vectors: These vectors are allowed to infect the cell, transferring the recombinant DNA into the host.

Processes of Recombinant DNA Technology

Recombinant DNA technology involves a series of sequential steps:

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Ligation of the DNA fragment into a vector.
5. Transferring the recombinant DNA into the host.
6. Culturing the host cells on a large scale.
7. Extraction of the desired product.

Isolation of the Genetic Material (DNA)

Nucleic acid, primarily deoxyribonucleic acid (DNA), is the genetic material in most organisms.

• To cut DNA with restriction enzymes, it must be in a pure form, free from other macromolecules.
• Cells must be broken open to release DNA along with other macromolecules like RNA, proteins, polysaccharides, and lipids.
• This is achieved by treating cells/tissues with specific enzymes:
  • Lysozyme for bacterial cells.
  • Cellulase for plant cells.
  • Chitinase for fungal cells.
• RNA can be removed with ribonuclease, and proteins with protease.
• Finally, purified DNA precipitates out as fine threads upon the addition of chilled ethanol.

Cutting of DNA at Specific Locations

• Restriction enzyme digestions are performed by incubating purified DNA molecules with the specific restriction enzyme under optimal conditions.
• Agarose gel electrophoresis is used to check the progression of the digestion.
• The cut ‘gene of interest’ from the source DNA and the cut vector are mixed, and ligase is added to prepare the recombinant DNA.

Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction.

• It synthesises multiple copies (amplifies) of the gene or DNA of interest in vitro.
• The reaction uses two sets of primers (small chemically synthesised oligonucleotides complementary to DNA regions) and the enzyme DNA polymerase.
• The enzyme extends the primers using provided nucleotides and the genomic DNA as a template.
• Repeated cycles of DNA replication can amplify the DNA segment approximately a billion times.
• This amplification is made possible by a thermostable DNA polymerase (isolated from Thermus aquaticus), which remains active during the high-temperature denaturation of double-stranded DNA.
• The amplified fragment can then be ligated with a vector for further cloning.

Insertion of Recombinant DNA into the Host Cell/Organism

Once made competent, recipient cells take up the surrounding DNA.

• For example, if recombinant DNA bearing a gene for ampicillin resistance is transferred into E. coli cells, the host cells become ampicillin-resistant.
• When these transformed cells are spread on agar plates containing ampicillin, only the transformed cells (with resistance) will grow, while untransformed cells will die. The ampicillin resistance gene thus acts as a selectable marker.

Obtaining the Foreign Gene Product

The ultimate aim of most recombinant technologies is to produce a desirable protein.

• When alien DNA is inserted into a cloning vector and transferred into a host cell (bacterial, plant, or animal), it multiplies, and the foreign gene is expressed under appropriate conditions.
• A protein encoded by a foreign gene expressed in a heterologous host is called a recombinant protein.
• Cultures of cells harbouring cloned genes can be grown on a small scale in the laboratory for extracting and purifying the desired protein using various separation techniques.

Large-Scale Production:

• Small volume cultures yield insufficient product quantities, necessitating bioreactors for large-scale production (100-1000 litres of culture).
• Bioreactors are vessels where raw materials are biologically converted into specific products, enzymes, etc., using microbial, plant, animal, or human cells.
• They provide optimal growth conditions, including temperature, pH, substrate, salts, vitamins, and oxygen, to achieve the desired product.
• Stirred-tank bioreactors are commonly used, designed cylindrically or with a curved base for efficient mixing and oxygen availability.
• They include an agitator, oxygen delivery system, foam control, temperature control, pH control, and sampling ports for periodic withdrawal of culture samples.
• Continuous culture systems in bioreactors drain used medium and add fresh medium, maintaining cells in their physiologically most active (log/exponential) phase, leading to larger biomass and higher protein yields.

Downstream Processing

After the biosynthetic stage (where the product is made), the product undergoes a series of processes before it is ready for marketing as a finished product; these are collectively known as downstream processing.

• This includes separation and purification.
• The product must be formulated with suitable preservatives.
• Such formulations undergo thorough clinical trials, similar to drugs.
• Strict quality control testing is also required for each product.
• Downstream processing and quality control testing procedures vary depending on the specific product.