Biotechnology: Principles and Processes

Key Concepts

Definition of Biotechnology

Techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

European Federation of Biotechnology (EFB) Definition: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Principles of Biotechnology

Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA) and introduce them into host organisms to change the phenotype.
Bioprocess Engineering: Maintenance of sterile ambience to enable the growth of only desired microbes/cells in large quantities for products like vaccines and enzymes.

Tools of Recombinant DNA Technology

Restriction Enzymes (Molecular Scissors):

First Restriction Enzyme Hind II was the first restriction endonuclease to be discovered. Its functioning depended on a specific DNA nucleotide sequence of six base pairs.

Hind II: First restriction endonuclease isolated; cuts DNA at specific 6-bp recognition sequences.
Nomenclature: e.g., EcoRI (E = Escherichia, co = coli, R = RY13 strain, I = order of isolation).
Types: Exonucleases (remove nucleotides from ends) and Endonucleases (cut at specific positions within DNA).
Palindromes: DNA sequences that read the same on two strands in the 5'→3' direction.
Sticky Ends: Overhanging single-stranded portions that facilitate ligation.

Cloning Vectors:
- Plasmids and Bacteriophages are common vectors.
- Features: Origin of replication (ori), Selectable markers (antibiotic resistance genes like ampR, tetR), and Cloning sites.
- Insertional Inactivation: e.g., Inactivating β-galactosidase gene results in white colonies (recombinants) vs blue colonies (non-recombinants).
- Vectors for Plants/Animals: Agrobacterium tumifaciens (Ti-plasmid) for plants and disarmed Retroviruses for animals.
Competent Host:
- Cells made competent by divalent cation treatment (e.g., Calcium) followed by heat shock (42°C).
- Other methods: Micro-injection, Biolistics (Gene gun) using gold/tungsten particles.

Processes of Recombinant DNA Technology

Isolation of DNA: Breaking cells with enzymes (Lysozyme, Cellulase, Chitinase) and precipitating DNA with chilled ethanol.
Fragmentation: Digestion with restriction enzymes.
Separation: Gel Electrophoresis (DNA moves toward anode; smaller fragments move farther).

Ethidium Bromide Safety DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation. You cannot see pure DNA fragments in the visible light.

Amplification (PCR):
- Polymerase Chain Reaction (PCR) is used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence in vitro.
- Three main steps:
  - Denaturation ( $94^\circ\text{C}\text{–}96^\circ\text{C}$ ): Double-stranded target DNA is heated to separate the two strands by breaking the hydrogen bonds.
  - Annealing ( $50^\circ\text{C}\text{–}65^\circ\text{C}$ ): Primers (short synthetic oligonucleotides) are allowed to hybridize to their complementary sequences on the single-stranded DNA templates.
  - Extension ( $72^\circ\text{C}$ ): The thermostable Taq Polymerase (isolated from hot-spring bacterium Thermus aquaticus) adds dNTPs to the 3'-OH end of the primers, extending the strands along the templates.
Ligation: Joining gene of interest and vector using DNA Ligase.
Transformation: Introducing recombinant DNA into the host.
Large-scale Production:
- Bioreactors: Large vessels (100-1000 L) providing optimal growth conditions (temp, pH, O2).
- Stirred-tank Bioreactor: Facilitates mixing and aeration.
Downstream Processing: Separation, purification, formulation with preservatives, and quality control testing before marketing.

Key Concepts

Techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

European Federation of Biotechnology (EFB) Definition: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA) and introduce them into host organisms to change the phenotype.

Bioprocess Engineering: Maintenance of sterile ambience to enable the growth of only desired microbes/cells in large quantities for products like vaccines and enzymes.

Restriction Enzymes (Molecular Scissors):

First Restriction Enzyme Hind II was the first restriction endonuclease to be discovered. Its functioning depended on a specific DNA nucleotide sequence of six base pairs.

Hind II: First restriction endonuclease isolated; cuts DNA at specific 6-bp recognition sequences.

Nomenclature: e.g., EcoRI (E = Escherichia, co = coli, R = RY13 strain, I = order of isolation).

Types: Exonucleases (remove nucleotides from ends) and Endonucleases (cut at specific positions within DNA).

Palindromes: DNA sequences that read the same on two strands in the 5'→3' direction.

Sticky Ends: Overhanging single-stranded portions that facilitate ligation.

Cloning Vectors:

Plasmids and Bacteriophages are common vectors.
Features: Origin of replication (ori), Selectable markers (antibiotic resistance genes like ampR, tetR), and Cloning sites.
Insertional Inactivation: e.g., Inactivating β-galactosidase gene results in white colonies (recombinants) vs blue colonies (non-recombinants).
Vectors for Plants/Animals: Agrobacterium tumifaciens (Ti-plasmid) for plants and disarmed Retroviruses for animals.

Competent Host:

Cells made competent by divalent cation treatment (e.g., Calcium) followed by heat shock (42°C).
Other methods: Micro-injection, Biolistics (Gene gun) using gold/tungsten particles.

Isolation of DNA: Breaking cells with enzymes (Lysozyme, Cellulase, Chitinase) and precipitating DNA with chilled ethanol.

Fragmentation: Digestion with restriction enzymes.

Separation: Gel Electrophoresis (DNA moves toward anode; smaller fragments move farther).

Amplification (PCR):

Polymerase Chain Reaction (PCR) is used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence in vitro.

Three main steps:
- Denaturation ( $94^\circ\text{C}\text{–}96^\circ\text{C}$ ): Double-stranded target DNA is heated to separate the two strands by breaking the hydrogen bonds.
- Annealing ( $50^\circ\text{C}\text{–}65^\circ\text{C}$ ): Primers (short synthetic oligonucleotides) are allowed to hybridize to their complementary sequences on the single-stranded DNA templates.
- Extension ( $72^\circ\text{C}$ ): The thermostable Taq Polymerase (isolated from hot-spring bacterium Thermus aquaticus) adds dNTPs to the 3'-OH end of the primers, extending the strands along the templates.

Ligation: Joining gene of interest and vector using DNA Ligase.

Transformation: Introducing recombinant DNA into the host.

Large-scale Production:

Bioreactors: Large vessels (100-1000 L) providing optimal growth conditions (temp, pH, O2).
Stirred-tank Bioreactor: Facilitates mixing and aeration.

Downstream Processing: Separation, purification, formulation with preservatives, and quality control testing before marketing.

Biotechnology: Principles and Processes

Biotechnology: Principles and Processes

Key Concepts

Definition of Biotechnology

Principles of Biotechnology

Tools of Recombinant DNA Technology

Processes of Recombinant DNA Technology

On this page

BioBot

Biotechnology: Principles and Processes

Biotechnology: Principles and Processes

Key Concepts

Definition of Biotechnology

Principles of Biotechnology

Tools of Recombinant DNA Technology

Processes of Recombinant DNA Technology

On this page