Biotechnology: Principles and Processes

Learning Outcomes:

1. Understand the basic principles and processes of biotechnology.
2. Identify the tools used in recombinant DNA technology.
3. Grasp the methods involved in creating genetically modified organisms.
4. Learn about DNA isolation, gene cloning, and recombinant DNA construction.

Principles of Biotechnology

Biotechnology, in its modern form, involves using living organisms or their enzymes to produce useful products. It encompasses processes like making curd, bread, or wine, which rely on microbes. However, biotechnology now mainly refers to methods involving genetically modified organisms. Techniques like in vitro fertilization, DNA vaccine development, or gene therapy are prominent examples.

Important Concept:
Modern biotechnology is grounded in two key techniques: genetic engineering and bioprocess engineering.

1. Genetic Engineering: Refers to modifying the genetic material (DNA or RNA) of organisms to alter their traits.
2. Bioprocess Engineering: Involves maintaining a sterile environment to enable the growth of desired cells or microbes to produce products such as antibiotics or enzymes.

Tools of Recombinant DNA Technology

Several tools are essential for recombinant DNA technology. They enable the creation of genetically modified organisms. These tools include restriction enzymes, ligases, vectors, and host organisms.

Restriction Enzymes

Restriction enzymes are crucial for cutting DNA at specific sequences. They were first discovered in 1963. Hind II was the first to be characterized for its ability to cut at a specific sequence of six base pairs. Today, over 900 restriction enzymes are known, each cutting at specific sites. The naming convention involves the genus, species, and strain of the bacteria they are derived from.

1. Restriction Endonucleases: These cut DNA at specific points, recognizing a palindromic sequence.
2. Exonucleases and Endonucleases: Exonucleases remove nucleotides from DNA ends, while endonucleases cut at internal sites.
3. Sticky Ends: When restriction enzymes cut, they leave overhanging segments called sticky ends, which facilitate the joining of DNA fragments.

Important Concept:
Palindromic sequences in DNA read the same in opposite directions, such as 5′ – GAATTC – 3′ and 3′ – CTTAAG – 5′.

Cloning Vectors

Vectors are carriers for foreign DNA fragments. They replicate independently in bacterial cells, and commonly used vectors include plasmids and bacteriophages.

1. Origin of Replication (ori): This sequence controls the replication of DNA linked to it, and vectors with high copy numbers are preferred for cloning.
2. Selectable Markers: These help identify transformants (cells that have successfully taken up foreign DNA) by providing antibiotic resistance.
3. Cloning Sites: Vectors need restriction sites to facilitate the insertion of foreign DNA, such as the BamH I site in the pBR322 plasmid.

Note:
The insertion of foreign DNA into a cloning vector at a site like BamH I inactivates one antibiotic resistance gene while retaining another, enabling the selection of recombinants.

Competent Host for Transformation

DNA is hydrophilic, so it cannot pass through cell membranes without assistance. Bacterial cells are made competent to take up DNA by treatment with calcium ions. The process involves heat shock to enhance DNA uptake.

1. Micro-injection: DNA is directly injected into the nucleus of animal cells.
2. Biolistics (Gene Gun): Gold or tungsten particles coated with DNA are shot into plant cells.
3. Disarmed Pathogens: Pathogens like Agrobacterium tumifaciens are used to transfer DNA into plant cells.

Processes of Recombinant DNA Technology

Recombinant DNA technology follows a series of well-defined steps, from DNA isolation to gene amplification and insertion into host organisms. Each step is essential for creating and multiplying recombinant DNA.

Isolation of DNA

DNA, the genetic material in all organisms, must be extracted in its pure form. Cells are broken open using enzymes like lysozyme (for bacteria), cellulase (for plants), or chitinase (for fungi). Once isolated, proteins are removed using proteases, and RNA is degraded with ribonucleases. Chilled ethanol helps precipitate DNA, which is collected as fine threads.

1. Purification of DNA: DNA is separated from other macromolecules and visualized after precipitation.

Important Concept:
Pure DNA is essential for subsequent cutting and manipulation steps in recombinant DNA technology.

Cutting of DNA at Specific Sites

DNA is cut using restriction enzymes under optimal conditions. This creates fragments, which are separated using gel electrophoresis. DNA fragments, which are negatively charged, move towards the anode in an electric field. The smaller fragments move faster through the agarose gel.

1. Elution: The separated DNA fragments are extracted from the gel for further use.
2. Recombinant DNA Formation: The gene of interest is ligated into the cut vector, resulting in recombinant DNA.

Gene Amplification Using PCR

Polymerase Chain Reaction (PCR) amplifies the gene of interest by synthesizing multiple copies of DNA. PCR uses two primers and the enzyme DNA polymerase to replicate DNA. The process involves three main steps: denaturation, primer annealing, and extension.

1. Thermostable DNA Polymerase: Derived from the bacterium Thermus aquaticus, this enzyme remains active during the high-temperature phases of PCR.
2. Amplification: PCR can produce billions of copies of DNA, which can be used for further cloning or analysis.

Table: Comparison of Gene Amplification Techniques

Method	Purpose	Enzyme Used	Amplification Result
PCR	Amplify specific DNA	Thermostable DNA polymerase	Billions of DNA copies
Cloning	Insert gene into vector	DNA Ligase	Multiple clones in host

Insertion of Recombinant DNA into Host Cells

After amplification, recombinant DNA is introduced into host cells, which are made competent. For example, if the recombinant DNA confers ampicillin resistance, the transformed cells will survive on ampicillin-containing plates, allowing selection of transformed cells.

1. Transformation: The uptake of recombinant DNA by host cells.
2. Selectable Markers: Help distinguish transformed cells from non-transformed ones, e.g., ampicillin resistance.

Production of Foreign Gene Products

Once inside a host, the foreign DNA gets expressed to produce a desirable protein. The cells containing the recombinant DNA are cultured on a large scale. This is achieved through bioreactors, which provide optimal growth conditions.

1. Recombinant Protein Production: The ultimate goal is to produce a functional protein on a large scale.
2. Continuous Culture System: Ensures a steady supply of nutrients to the cells while removing waste products, optimizing protein production.

Table: Bioreactor Types and Features

Bioreactor Type	Description	Features
Stirred-Tank Bioreactor	Cylindrical with curved base	Stirring system for even mixing
Sparged Bioreactor	Air bubbled through the culture	Better oxygen availability

Downstream Processing

After biosynthesis, the product must be purified and processed. This includes separation and purification steps, collectively known as downstream processing. The purified product is then formulated with preservatives and subjected to quality control.

1. Formulation: The product is stabilized with preservatives.
2. Quality Control: Ensures that the final product meets safety and efficacy standards.

Summary of Key Concepts

Note:
Understanding the intricacies of DNA manipulation has led to significant advancements in biotechnology. The creation of recombinant DNA, gene cloning, and the ability to amplify and express foreign genes have revolutionized modern science and medicine.

MCQ:

What is the purpose of the origin of replication (ori) in a cloning vector?
A) To allow the foreign DNA to replicate in the host cells
B) To introduce antibiotic resistance
C) To insert foreign DNA into host cells
Answer: A