Biotechnology : Principles And Processes

Principles Of Biotechnology

Biotechnology is the use of living organisms or systems to make products or processes for a specific use. EFB (European Federation of Biotechnology) defines it as "the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services".

Traditional biotechnology involves processes like making curd, bread, or wine, which rely on naturally occurring microbial processes. Modern biotechnology, often referred to as genetic engineering, involves manipulating genetic material (DNA) to alter organisms or produce new substances.

Core Techniques of Modern Biotechnology:

Two main techniques that paved the way for modern biotechnology are:

Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA) and introduce these into host organisms, thus changing the phenotype of the host organism.
- It involves the creation of recombinant DNA (rDNA), where DNA from two different sources (e.g., a desired gene from one organism and a vector DNA) is combined.
- Recombinant DNA technology allows the transfer of desirable genes across species barriers.
Bioprocess Engineering: Maintaining sterile conditions in chemical engineering processes to enable the growth of only the desired microbe or eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, hormones, etc.
- This involves using fermentors or bioreactors under controlled conditions (temperature, pH, nutrient supply, oxygen).

Creation of Recombinant DNA:

The first recombinant DNA molecule was constructed by Stanley Cohen and Herbert Boyer in 1972. They achieved this by:

Isolating an antibiotic resistance gene from a plasmid of *Salmonella typhimurium*.
Cutting this gene out using restriction enzymes.
Cutting a specific plasmid DNA (acting as a vector) using the same restriction enzyme.
Joining the antibiotic resistance gene to the plasmid DNA using the enzyme DNA ligase. This created a recombinant plasmid.
Transferring this recombinant plasmid into *E. coli* bacteria.
The *E. coli* bacteria became resistant to the antibiotic, demonstrating that the introduced gene was successfully expressed.

This experiment demonstrated the key steps of recombinant DNA technology: identifying and isolating a gene, cutting DNA using restriction enzymes, joining DNA fragments using ligase, and transferring recombinant DNA into a host.

Diagram illustrating the process of creating recombinant DNA by joining a foreign gene with a vector plasmid

*(Image shows a diagram illustrating a foreign DNA fragment being cut by a restriction enzyme, a plasmid vector being cut by the same enzyme, and the foreign DNA fragment being ligated into the plasmid to form a recombinant plasmid)*

Advantages of Genetic Engineering over Traditional Hybridisation:

Traditional hybridisation in plant/animal breeding often leads to the introduction of undesirable genes along with desirable genes, as crosses occur between whole organisms.
Genetic engineering allows the introduction of only the desirable gene(s) into the target organism, overcoming the limitations of traditional methods and allowing precise manipulation of genetic traits.
It also allows the transfer of genes between unrelated species (across sexual barriers).

The principles of genetic engineering and bioprocess engineering form the foundation of modern biotechnology, enabling the development of various applications in medicine, agriculture, and industry.

Tools Of Recombinant Dna Technology

Creating recombinant DNA and introducing it into a host organism requires several key tools. These are the 'molecular tools' used in genetic engineering.

The main tools of recombinant DNA technology are:

Restriction enzymes
Cloning vectors
Competent host
DNA ligase (discussed in replication, used for joining DNA fragments)
Polymerase enzymes (like Taq polymerase used in PCR)

Restriction Enzymes

These are enzymes that cut DNA at specific recognition sequences. They are also called 'molecular scissors' or 'chemical scalpels'.
Restriction enzymes are found naturally in bacteria, where they function as part of a defense mechanism against viruses (they cut viral DNA).
The first restriction endonuclease was Hind II.
Restriction enzymes belong to a class of enzymes called nucleases. Nucleases are of two types:
- Exonucleases: Remove nucleotides from the ends of the DNA molecule.
- Endonucleases: Cut at specific positions within the DNA molecule. Restriction endonucleases are this type.

Recognition Sites:

Each restriction endonuclease recognises a specific DNA sequence, usually a 4-8 base pair sequence.

These recognition sequences are often palindromic, meaning the sequence reads the same forwards and backward on the two complementary strands.
Example: EcoRI recognises the sequence $5'-GAATTC-3'$ on one strand, which is $3'-CTTAAG-5'$ on the complementary strand. Reading from 5' to 3' on both strands gives GAATTC.

Cutting Pattern:

Restriction enzymes cut the DNA double helix at specific sites within the recognition sequence.

Some enzymes cut both strands at the same position, generating blunt ends.

Many enzymes cut the strands at different positions, generating sticky ends (or cohesive ends). Sticky ends are short single-stranded overhangs.
Example: EcoRI cuts between G and A on both strands within the GAATTC site, creating sticky ends.

Sticky ends are very useful in recombinant DNA technology because they can form hydrogen bonds with complementary sticky ends of other DNA fragments cut with the same enzyme. This facilitates the joining of DNA fragments by DNA ligase.

(Image shows diagrams illustrating a DNA molecule being cut by restriction enzymes at recognition sites, showing the formation of sticky ends (staggered cut) and blunt ends (straight cut))

Nomenclature of Restriction Enzymes:
The naming follows a standard convention:

The first letter comes from the genus of the bacterium (capitalised).

The next two letters come from the species of the bacterium (lowercase).

The fourth letter comes from the strain of the bacterium (optional).

The Roman numeral indicates the order of discovery from that strain.

Example: EcoRI comes from Escherichia coli RY13 (E - Escherichia, co - coli, R - strain RY, I - first enzyme isolated).

Cloning Vectors

A vector is a DNA molecule that can carry a foreign DNA segment and replicate inside a host cell. Vectors are used to introduce a gene of interest into a host organism and amplify it.
Commonly used vectors include plasmids (small circular extra-chromosomal DNA in bacteria), bacteriophages (viruses that infect bacteria), and artificial chromosomes (BACs, YACs).

Features Required in a Cloning Vector:
An ideal cloning vector should have the following features:

Origin of replication (ori): A specific DNA sequence that allows the vector to replicate autonomously within the host cell. This sequence also controls the copy number of the vector.

Selectable marker: A gene that helps in identifying and selecting transformed host cells (cells that have taken up the vector) and eliminating non-transformants. Examples: Genes encoding resistance to antibiotics (e.g., ampicillin, tetracycline).

Cloning sites (Restriction sites): Recognition sites for various restriction enzymes, where the foreign DNA can be ligated. Presence of single recognition sites for different restriction enzymes within the selectable marker or a reporter gene facilitates selection of recombinants.

Reporter gene (optional but useful): A gene whose expression can be easily monitored (e.g., gene coding for an enzyme like $\beta$-galactosidase, which can convert a substrate into a coloured product). Insertion of foreign DNA into this gene inactivates its expression (insertional inactivation), allowing easy distinction between recombinants and non-recombinants.

Example of a Cloning Vector: pBR322:
pBR322 is a widely used artificial plasmid vector constructed in the laboratory. It has:

An ori sequence.

Two selectable markers: genes for resistance to ampicillin ($amp^R$) and tetracycline ($tet^R$).

Multiple unique cloning sites within these resistance genes and other regions (e.g., HindIII, EcoRI, BamHI, SalI, PvuI, PvuII, ClaI).

Insertion of foreign DNA at the BamHI site (within $tet^R$ gene) makes the recombinant plasmid lose tetracycline resistance but retain ampicillin resistance. Non-recombinants will have resistance to both antibiotics. This allows selection of recombinants by plating on media containing ampicillin (both grow), then transferring colonies to a medium containing tetracycline (only non-recombinants grow). Recombinants are those that grew on ampicillin but not on tetracycline.

(Image shows a circular map of pBR322 indicating ori, ampR gene, tetR gene, and locations of various restriction enzyme sites within these genes or outside)

Competent Host (For Transformation With Recombinant Dna)

After creating recombinant DNA, it needs to be introduced into a living host cell where it can replicate and express the gene.
Usually, bacterial cells are used as hosts (e.g., *E. coli*). However, DNA is a hydrophilic molecule and cannot easily pass through the cell membrane.
The host cells must be made 'competent' to take up foreign DNA. This involves making the cell membrane permeable to DNA.

Methods to make Host Cells Competent:

Chemical treatment: Treating bacterial cells with specific chemicals, like a divalent cation (e.g., calcium chloride, $CaCl_2$), which creates pores in the cell wall and membrane.

Heat shock: After chemical treatment, the cells are incubated with recombinant DNA on ice, then subjected to a brief heat pulse (heat shock at $42^\circ C$), and then placed back on ice. This helps the DNA enter the cells.

Microinjection: Directly injecting recombinant DNA into the nucleus of an animal cell using a fine needle.

Biolistics (Gene gun): Coating DNA onto microscopic gold or tungsten particles and shooting them into plant cells using a high-velocity 'gene gun'.

Disarmed pathogens: Using modified viruses or bacteria that are rendered non-pathogenic but can still deliver the foreign DNA into the host cell.

Once the recombinant DNA is inside the host cell, the process of transformation (genetic alteration of a cell by uptake of foreign DNA) is complete. The transformed host cell can then be grown to multiply the recombinant DNA and express the foreign gene.

Processes Of Recombinant Dna Technology

Recombinant DNA technology involves a series of steps to create a genetically modified organism or produce a desired protein. These steps are performed in a specific sequence.

The main processes of recombinant DNA technology are:

Isolation of the genetic material (DNA).
Cutting of DNA at specific locations.
Amplification of gene of interest (optional, but often needed).
Insertion of recombinant DNA into the host cell/organism.
Obtaining the foreign gene product.
Downstream processing.

Isolation Of The Genetic Material (Dna)

The first step is to obtain pure DNA from the donor organism's cells.
Cells are treated with enzymes to break down cell structures:
- Cell wall: Cellulase (for plant cells), lysozyme (for bacteria), chitinase (for fungi).
- Cell membrane: Digested using enzymes and detergents.
RNA is removed by treating with ribonuclease (RNase).
Proteins are removed by treating with proteases.
Other molecules are also removed.
Finally, pure DNA is precipitated by adding chilled ethanol. DNA precipitates as fine threads that can be collected by spooling (winding the DNA onto a glass rod).

Diagram illustrating the isolation of DNA (cell lysis, removal of RNA/proteins, precipitation with ethanol, spooling)

*(Image shows a simplified diagram illustrating the steps of DNA isolation, including cell lysis, treatment with enzymes, and precipitation/spooling of DNA)*

Cutting Of Dna At Specific Locations

The purified DNA from the donor organism and the vector DNA are cut at specific sites using restriction enzymes.
It is crucial to use the same restriction enzyme to cut both the donor DNA (containing the gene of interest) and the vector DNA. This ensures that they generate complementary sticky ends (if the enzyme produces sticky ends), which are essential for ligation.
The gene of interest is a specific segment of the donor DNA.
Gel electrophoresis is used to check the success of the cutting reactions and to separate DNA fragments of different sizes. The gene of interest can be identified and isolated from the gel based on its size.

Amplification Of Gene Of Interest Using Pcr

Often, the amount of gene of interest isolated is very small. For subsequent steps, it is necessary to amplify (make multiple copies of) the gene.
Polymerase Chain Reaction (PCR) is a technique used to synthesise multiple copies of the gene of interest in vitro (in a test tube). PCR was developed by Kary Mullis.
PCR requires:
- Desired DNA template: Containing the gene of interest.
- Primers: Short, synthetic oligonucleotide sequences complementary to the regions flanking the gene of interest. Two primers are needed, one for each strand, facing each other.
- Deoxyribonucleotides: dNTPs (dATP, dGTP, dCTP, dTTP) as building blocks.
- DNA polymerase: A thermostable DNA polymerase is used, such as Taq polymerase, isolated from the bacterium *Thermus aquaticus*, which can tolerate high temperatures.

Steps of PCR (Cycles):
Each cycle of PCR involves three steps:

Denaturation: The DNA template is heated to a high temperature ($94-96^\circ C$) to separate the two strands.

Annealing: The temperature is lowered ($50-65^\circ C$) to allow the primers to bind (anneal) to their complementary sequences on the separated template strands.

Extension (Polymerisation): The temperature is raised to the optimal temperature for the DNA polymerase ($72^\circ C$ for Taq polymerase). DNA polymerase adds nucleotides to the 3' end of the primers, synthesising new DNA strands complementary to the templates.

These three steps are repeated for 20-40 cycles in a thermal cycler. Each cycle doubles the amount of DNA. After 'n' cycles, the amount of DNA is $2^n$ times the initial amount. This allows for rapid amplification of the gene of interest.

(Image shows a diagram illustrating a few cycles of PCR, showing DNA denaturation, primer annealing, and extension by DNA polymerase)

Ligation of DNA Fragments:

The gene of interest (donor DNA fragment) and the cut vector DNA are mixed together.

The enzyme DNA ligase is added to join the foreign DNA fragment into the vector DNA, forming a recombinant DNA (rDNA) molecule. Complementary sticky ends facilitate this ligation.

Insertion Of Recombinant Dna Into The Host Cell/Organism

The rDNA molecule is introduced into a suitable host cell (e.g., bacterium, plant cell, animal cell) that has been made competent to take up the foreign DNA.
Methods used include chemical treatment and heat shock (for bacteria), microinjection (for animal cells), biolistics (for plant cells), or using disarmed pathogens (vectors).
Host cells that have successfully taken up the rDNA are called transformed cells.
Selection of transformed cells is done using selectable markers present on the vector (e.g., antibiotic resistance). For example, transformed bacteria containing the recombinant plasmid with ampicillin resistance will grow on a medium containing ampicillin, while non-transformed bacteria will die.
Distinguishing between recombinants (carrying the gene of interest) and non-recombinants (carrying only the vector without the insert) is done using reporter genes or insertional inactivation of selectable markers (as discussed in the pBR322 example).

Obtaining The Foreign Gene Product

Once the recombinant DNA is successfully introduced into a host cell, the goal is often to produce the protein encoded by the foreign gene (recombinant protein).
This involves the expression of the foreign gene in the host (transcription and translation).
The host cell containing the rDNA is grown in a suitable culture medium under optimal conditions.
If the foreign gene is expressed, the host cell will produce the desired protein.
For large-scale production, the host cells are grown in large fermentors (bioreactors).

Optimising Expression:

Expression vectors are designed to ensure high-level expression of the foreign gene (e.g., having strong promoters, regulatory sequences).

The conditions in the fermentor are controlled (temperature, pH, oxygen, nutrients) to optimise the growth of the host and the production of the protein.

Downstream Processing

After the desired gene product (protein) is produced, it needs to be purified and processed for market. This is called downstream processing.
It involves a series of steps:
- Separation: Separating the protein from the host cells and culture medium. This may involve cell lysis (if the protein is intracellular), filtration, centrifugation.
- Purification: Purifying the desired protein from other cellular components and proteins using techniques like chromatography.
- Formulation: Converting the purified protein into a suitable form for use (e.g., adding preservatives).
- Quality control testing: Ensuring the product meets purity, safety, and efficacy standards.
- Clinical trials: For pharmaceutical products.
Downstream processing varies for different products.

These steps constitute the core process of recombinant DNA technology, enabling the creation of genetically modified organisms and the production of valuable proteins for various applications.