Biotechnology Principles And Processes: NCERT Concepts, Recombinant DNA Technology And Bioreactors

Traditional biotechnology uses living organisms or enzymes to make useful products such as curd, bread, wine, and beer. These processes use the natural activities of microorganisms to produce food and other useful substances.

Modern biotechnology also focuses on genetic modification and large-scale production. It includes techniques such as genetic engineering, gene cloning, in vitro fertilisation (IVF), vaccine development, recombinant protein production, and controlled bioprocessing for industrial applications.

Meaning And Scope Of Biotechnology

Biotechnology involves using living organisms, cells, enzymes, or biological molecules to develop useful products and services. It includes traditional methods based on natural biological activities and modern methods that use genetic modification and controlled production techniques.

Traditional And Modern Biotechnology

Traditionally, biotechnology meant using living organisms or enzymes derived from organisms to produce useful products. Examples include:

• Conversion of milk into curd by Lactobacillus

• Wine production by Saccharomyces cerevisiae

• Beer production from malted cereals or rice

• Bread production through microbial fermentation

• Alcohol production through anaerobic respiration

Modern biotechnology has a more restricted meaning. It generally requires:

1. Use of a genetically modified organism

2. Production on a large scale

A genetically modified organism has deliberately altered genetic material. This alteration may involve adding a gene, modifying an existing gene, or introducing genetic material from another organism.

Biotechnology also includes techniques such as in vitro fertilisation, gene synthesis, DNA and RNA vaccine development, and correction of defective genes.

IVF And GIFT

In in vitro fertilisation (IVF), sperm and ovum fuse in a laboratory, outside the body. Since fertilisation occurs in vitro, IVF is included under biotechnology.

In gamete intrafallopian transfer (GIFT), the ovum is transferred into the fallopian tube. Fertilisation occurs naturally inside the body. Therefore, GIFT is not included in biotechnology in this context.

EFB Definition

The European Federation Of Biotechnology defines biotechnology as the integration of natural sciences with organisms, cells, parts of cells, molecules, or analogous systems to produce products and services.

Two Core Techniques Of Modern Biotechnology

Modern biotechnology depends mainly on:

1. Genetic Engineering

2. Bioprocess Engineering

Genetic engineering creates the producer; bioprocess engineering grows the producer safely and in bulk.

Genetic Engineering

Genetic engineering involves altering the chemistry of genetic material, introducing it into a host, and changing the phenotype of that host.

It can:

• Transfer a desired gene into a host

• Change the host genotype

• Change the host phenotype

• Enable production of a desired product

Bioprocess Engineering

Genetic modification alone is not enough for industrial production. The selected organism must be grown in large quantities without contamination.

Bioprocess engineering involves maintaining sterile conditions so that only the desired microorganism or eukaryotic cell grows and produces the required product.

Sexual Reproduction And Traditional Hybridisation

Sexual reproduction combines genetic material from two individuals and produces variation. It creates new genetic combinations, some of which may be beneficial.

Asexual reproduction preserves the existing genetic constitution and produces genetically similar offspring.

Traditional hybridisation can combine desirable traits, but it may also transfer unwanted genes. Genetic engineering solves this problem by transferring only the selected gene of interest.

Foreign DNA, Origin Of Replication And Gene Cloning

Foreign DNA does not automatically replicate inside a host. It must be associated with an origin of replication.

Foreign DNA can multiply in two ways:

1. It may integrate into the host chromosome and replicate with it.

2. It may be linked to a DNA molecule containing its own origin of replication.

An origin of replication is the DNA sequence from which replication begins.

Gene cloning is the production of multiple identical copies of a particular DNA molecule or gene. The number of copies can be written as:

Number Of Copies = 2 Raised To N

Here, N represents the number of replication cycles.

Plasmids As Vectors

A bacterial cell contains genomic DNA and may also contain a plasmid. A plasmid is an extra-chromosomal, circular, self-replicating DNA molecule.

A plasmid can:

1. Be isolated from a bacterium

2. Receive the gene of interest

3. Enter a host bacterium

4. Replicate inside the host

5. Produce multiple copies of the inserted gene

A plasmid used to carry foreign DNA is called a vector.

Recombinant DNA And Molecular Tools

Recombinant DNA is formed by joining DNA fragments from different sources. It usually contains the gene of interest and a vector sequence with an origin of replication.

Two essential molecular tools are:

• Restriction Endonuclease: Acts as molecular scissors and cuts DNA at specific recognition sites.

• DNA Ligase: Joins DNA fragments by sealing phosphodiester bonds.

Restriction enzymes commonly recognise palindromic sequences. These sequences read the same on both DNA strands when read in the same five-prime to three-prime direction.

EcoRI recognises the sequence GAATTC and cuts between G and A. This produces sticky ends, also called cohesive or staggered ends.

Sticky ends contain complementary overhangs that pair through hydrogen bonding. This makes it easier to align the gene of interest with the vector before DNA ligase permanently joins them.

Gel Electrophoresis And DNA Isolation

Restriction digestion produces several DNA fragments. The fragment containing the gene of interest is separated using agarose gel electrophoresis.

DNA is negatively charged, so it moves towards the positive electrode, or anode. Separation occurs according to size:

• Smaller DNA fragments move faster and travel farther.

• Larger DNA fragments move slowly and remain closer to the wells.

Agarose acts as a molecular sieve.

The gel is stained with ethidium bromide and viewed under ultraviolet light. DNA appears as bright orange bands. The required band is cut from the gel, and DNA is recovered through elution.

For linear DNA, the number of fragments is one more than the number of restriction sites. For circular DNA, the number of fragments equals the number of restriction sites.

Cloning Vectors And Selectable Markers

Cloning vectors carry the gene of interest into a suitable host cell and help produce multiple copies of the inserted DNA. A suitable vector must have specific features that support DNA replication, identification of host cells, and insertion of foreign DNA.

A suitable vector should contain:

1. Origin Of Replication

2. Selectable Marker

3. Cloning Site

The vector selected also depends on the size of the gene and the type of host.

Examples include:

• Plasmids for smaller genes in bacterial cells

• Bacteriophages for larger genes in bacterial hosts

• Disarmed pathogens for plant or animal cells

• Artificial chromosomes for very large DNA fragments

A selectable marker helps identify transformants and distinguish recombinants from non-recombinants. Common markers include resistance genes for ampicillin, tetracycline, chloramphenicol, and kanamycin.

PBR322 Selection

The artificial plasmid pBR322 contains an origin of replication, ampicillin-resistance gene, tetracycline-resistance gene, and restriction sites.

• Ampicillin resistance identifies transformants.

• Insertion of the gene of interest into the tetracycline-resistance gene disrupts tetracycline resistance.

• Recombinants survive ampicillin but die on tetracycline.

• Non-recombinants survive both antibiotics.

Insertional Inactivation And Blue-White Screening

In pUC18, the gene of interest is inserted into the lacZ gene.

• Recombinant colonies have disrupted lacZ and remain colourless on X-gal.

• Non-recombinant colonies have active lacZ and become blue.

This method is more convenient because the desired recombinant colony remains alive and can be selected directly.

Competent Host Cells And Transformation

DNA is hydrophilic, while the cell membrane is a hydrophobic lipid bilayer. Therefore, cells must be made competent to take up foreign DNA.

Methods include:

• Divalent cation treatment: Calcium ions create temporary pores.

• Electroporation: Short electrical pulses create membrane pores.

• Heat Shock: Bacterial cells undergo the sequence ice, heat, and ice.

Other host-specific methods are:

• Microinjection: Used for animal cells; DNA is injected into the nucleus.

• Biolistic Gun: Used for plant cells; DNA-coated gold or tungsten particles are shot into cells.

• Disarmed Pathogens: Modified pathogens deliver genes after harmful genetic material is removed.

PCR And DNA Amplification

Polymerase chain reaction (PCR) produces millions or billions of copies of a selected DNA segment in vitro.

PCR requires:

• Template DNA

• Two primers

• Deoxyribonucleotides

• DNA polymerase

The three steps are:

1. Denaturation: At about 95 degrees C, hydrogen bonds break and DNA strands separate.

2. Annealing: At about 42 degrees C, primers attach to complementary template regions.

3. Extension: DNA polymerase adds nucleotides in the five-prime to three-prime direction.

PCR uses Taq DNA polymerase, obtained from Thermus aquaticus. It is thermostable and remains active after repeated high-temperature cycles.

Bioreactors And Downstream Processing

A bioreactor is a large vessel used to grow genetically modified organisms and produce biological products under controlled conditions.

Important conditions include:

• Temperature

• pH

• Oxygen

• Nutrients

• Sterility

• Continuous mixing

A stirred-tank bioreactor contains a flat-bladed impeller, motor, foam breaker, pH-control port, sterile-air inlet, and medium inlet and outlet.

The impeller ensures uniform mixing and distributes oxygen throughout the culture. A foam breaker controls foam formation. Steam helps maintain sterility during port operations.

A sparged-type bioreactor supplies oxygen as bubbles. This increases the surface area for gas exchange and improves oxygen transfer.

After production, the product undergoes downstream processing:

1. Separation

2. Purification

3. Formulation

4. Packaging

5. Preservation, if required

6. Quality-control testing

7. Clinical trials for medicines

8. Marketing

Separation and purification are required for every product, while later downstream steps vary according to the product.

Common Mistakes to Avoid in Biotechnology Principles and Processes

Students should understand the differences between key biotechnology concepts to avoid confusion in NEET questions. The following mistakes are important to avoid during revision:

• Genetic Engineering vs Bioprocess Engineering: Genetic engineering modifies genetic material, while bioprocess engineering maintains suitable and sterile conditions for large-scale production.

• Restriction Enzymes vs DNA Ligase: Restriction endonucleases cut DNA at specific recognition sites, whereas DNA ligase joins DNA fragments by forming phosphodiester bonds.

• Transformants vs Recombinants: Transformants are host cells that take up foreign DNA. Recombinants are transformants that contain the desired recombinant DNA.

• Sticky Ends vs Blunt Ends: Sticky ends have complementary overhangs, while blunt ends have no single-stranded overhangs.

• pBR322 Selection: Recombinants with an insertion in the tetracycline-resistance gene survive on ampicillin but not tetracycline. Non-recombinants can survive on both antibiotics.

• Blue-White Screening: Recombinant colonies are usually colourless because the inserted DNA disrupts the lacZ gene. Non-recombinant colonies appear blue in the presence of X-gal under suitable conditions.

• PCR Steps: Remember the correct order: denaturation, annealing, and extension. Taq DNA polymerase is used because it can withstand repeated high-temperature cycles.

• DNA Movement in Gel Electrophoresis: DNA moves towards the positive electrode because it carries a negative charge. Smaller DNA fragments generally travel farther than larger fragments.

• Origin of Replication: Foreign DNA needs an origin of replication to replicate independently, unless it integrates into the host chromosome and replicates with it.

• Downstream Processing: It includes product separation and purification, followed by other steps such as formulation, packaging, and quality-control testing, depending on the product.

Understanding the principles and processes of biotechnology helps students revise key NCERT concepts, from recombinant DNA technology and PCR to bioreactors and downstream processing. Regular revision of these concepts and their differences can help strengthen NEET preparation.

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