Detailed Notes CHAPTER 9 -BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

Chapter 9- Biotechnology  : Principles And Processes

Introduction

1. Biotechnology Definition:

   • Traditional View: Involves using live organisms or their enzymes to create useful products and processes like curd, bread, or wine.
   • Modern View: Also includes processes with genetically modified organisms (GMOs) on a larger scale.

2. Examples of Biotechnological Processes:

   • Traditional Processes: Making curd, bread, or wine through microbial actions.
   • Modern Processes: In vitro fertilization (IVF), gene synthesis, DNA vaccine development, gene correction.

3. European Federation of Biotechnology (EFB) Definition:

   • Integration of Sciences: Merging natural sciences with living organisms, cells, parts, and molecular analogues.
   • Product and Service Focus: Aimed at creating products and services beneficial to humans.

4. Integration in Biotechnology:

   • Natural Sciences: Includes biology, chemistry, and genetics.
   • Organisms and Cells: Utilizes living organisms, cells, and their components.
   • Molecular Analogues: Involves artificial molecular constructs for biotechnological applications.

5. Biotechnological Products and Services:

   • Purpose: Creating products and services for human benefit.
   • Examples: Medicines, vaccines, agricultural improvements, industrial processes.

9.1 PRINCIPLES OF BIOTECHNOLOGY

1. Core Techniques of Modern Biotechnology:

   • Genetic Engineering: Modifying genetic material (DNA and RNA) and introducing it into host organisms to change their phenotype.
   • Bioprocess Engineering: Ensuring a sterile environment in engineering processes to grow desired microorganisms or eukaryotic cells for producing biotechnological products.

2. Conceptual Development of Genetic Engineering:

   • Advantages of Sexual Reproduction: Provides genetic variation, whereas asexual reproduction maintains genetic information.
   • Limitations of Traditional Hybridization: Inclusion of undesirable genes alongside desired ones.
   • Genetic Engineering Techniques: Overcome limitations by creating recombinant DNA, using gene cloning and transfer to introduce only desired genes without unwanted ones.

3. Fate of Transferred DNA:

   • Integration into Genome: Transferred DNA becomes part of the recipient organism’s chromosome, allowing it to replicate and be inherited in progeny cells.
   • Origin of Replication: Alien DNA needs to be linked to the origin of replication within a chromosome to replicate itself in the host organism.

4. Construction of Recombinant DNA:

   • Stanley Cohen and Herbert Boyer’s Work (1972): Linked an antibiotic resistance gene with a native plasmid of Salmonella typhimurium, creating the first recombinant DNA.
   • Molecular Scissors (Restriction Enzymes): Used to cut DNA at specific locations for genetic manipulation.
   • Plasmid as Vector: Acts like a carrier to transfer alien DNA into the host organism.
   • DNA Ligase: Enzyme that joins cut DNA ends, allowing the creation of recombinant DNA.

5. Genetic Modification Steps:

   • Identification of Desirable DNA: Finding DNA with desired genes.
   • Introduction into Host: Transferring the identified DNA into the host organism.
   • Maintenance and Transfer: Ensuring the introduced DNA replicates and transfers to progeny cells.

9.2 TOOLS OF RECOMBINANT DNA TECHNOLOGY

1. Restriction Enzymes:

   • Function: Cut DNA molecules at specific recognition sequences.
   • Importance: Essential for creating DNA fragments with desired genes for further manipulation.
   • Example: EcoRI, HindIII, BamHI are common restriction enzymes used in genetic engineering.

2. Polymerase Enzymes:

   • Function: Synthesize new DNA strands by adding nucleotides to a template DNA strand.
   • Importance: Used in PCR (Polymerase Chain Reaction) to amplify specific DNA regions and in DNA replication processes.
   • Examples: Taq polymerase, DNA polymerase I, DNA polymerase III.

3. Ligases:

   • Function: Join DNA fragments by catalyzing the formation of phosphodiester bonds between their ends.
   • Importance: Used to create recombinant DNA molecules by ligating DNA fragments with compatible ends.
   • Example: DNA ligase from Escherichia coli is commonly used in molecular biology techniques.

4. Vectors:

   • Definition: Carriers used to transfer foreign DNA into host cells.
   • Types: Plasmids, bacteriophages, artificial chromosomes (such as yeast artificial chromosomes or bacterial artificial chromosomes).
   • Importance: Provide a platform for DNA replication, storage, and expression of foreign genes in host organisms.
   • Features: Typically contain an origin of replication, selectable markers (e.g., antibiotic resistance genes), and cloning sites (restriction enzyme recognition sequences).

5. Host Organism:

   • Definition: Living organism that receives and replicates foreign DNA introduced by vectors.
   • Types: Bacteria (e.g., Escherichia coli), yeast, mammalian cells, plants, etc.
   • Importance: Provides the cellular machinery for gene expression, protein synthesis, and propagation of recombinant DNA.

9.2.1 Restriction Enzymes

1. Discovery and Function:

   • Discovered in 1963, restriction enzymes are enzymes that restrict the growth of bacteriophages in Escherichia coli.
   • One enzyme adds methyl groups to DNA, while the other, known as a restriction endonuclease, cuts DNA.

2. Hind II and Specificity:

   • Hind II, the first restriction endonuclease discovered, cuts DNA at a specific nucleotide sequence of six base pairs, known as the recognition sequence for Hind II.
   • Over 900 restriction enzymes have been isolated from various bacterial strains, each recognizing different recognition sequences.

3. Naming Convention:

   • Restriction enzymes are named based on the genus and species of the prokaryotic cell they were isolated from, following a specific naming convention.
   • For example, EcoRI comes from Escherichia coli RY 13, with the letter ‘R’ indicating the strain.

4. Types of Nucleases:

   • Restriction enzymes belong to a class of enzymes called nucleases, which are of two types: exonucleases and endonucleases.
   • Exonucleases remove nucleotides from DNA ends, while endonucleases make cuts at specific positions within the DNA.

5. Functioning of Restriction Endonucleases:

   • These enzymes recognize palindromic nucleotide sequences in DNA, which read the same in both directions.
   • They cut the DNA strands at specific points within the palindromic sequence, leaving single-stranded portions with sticky ends.

6. Palindromes in DNA:

   • DNA palindromes are sequences that read the same on both DNA strands, facilitating the action of restriction enzymes.
   • The cut sites of restriction enzymes are a little away from the center of the palindrome, creating single-stranded overhangs or sticky ends.

7. Application in Genetic Engineering:

   • Restriction enzymes are crucial in genetic engineering to create recombinant DNA molecules composed of DNA from different sources.
   • When DNA is cut by the same restriction enzyme, the resulting fragments have complementary sticky ends that can be joined using DNA ligase

   • Need for Compatible Restriction Enzymes:

     • To create recombinant vector molecules, both the vector and the source DNA must be cut with the same restriction enzyme. Otherwise, they won’t have complementary ends for joining.

1. Gel Electrophoresis:

   • Technique used to separate DNA fragments based on their size.
   • DNA fragments are negatively charged and move towards the anode (positive electrode) under an electric field in a gel matrix.

2. Agarose Gel:

   • Most commonly used matrix for gel electrophoresis, made from agarose, a natural polymer extracted from seaweeds.
   • Agarose gel acts as a sieve, allowing smaller DNA fragments to move faster and farther than larger ones.

3. Visualization of DNA Fragments:

   • DNA fragments are not visible to the naked eye, so they are stained with a compound like ethidium bromide.
   • Ethidium bromide-stained DNA bands are then visualized under UV light, appearing as bright orange bands on the gel.

4. Elution and Purification:

   • After separation, desired DNA fragments are cut out from the agarose gel in a process called elution.
   • Eluted DNA fragments are then purified and used for constructing recombinant DNA by joining them with cloning vectors.

9.2.2 Cloning Vectors

1. Ability to Replicate:

   • Plasmids and bacteriophages can replicate independently within bacterial cells, with varying copy numbers per cell.
   • Linking foreign DNA with these vectors allows multiplication equal to the vector’s copy number.

2. Features Required for Cloning Vectors:

   • Origin of Replication (ori): Initiates DNA replication, controlling the copy number of linked DNA. Vectors with high-copy origins are preferred for obtaining many copies of target DNA.
   • Selectable Marker: Identifies and eliminates non-transformants, allowing selective growth of transformants. Common markers include antibiotic resistance genes.
   • Cloning Sites: Few recognition sites for restriction enzymes to simplify gene cloning. Insertion of foreign DNA inactivates one antibiotic resistance gene, aiding in selecting recombinants.

3. Selection of Recombinants:

   • Inactivation of antibiotic resistance genes aids in selecting recombinants. Colonies growing on selective medium containing one antibiotic but not the other indicate successful cloning.
   • Alternative markers involve insertional inactivation of genes like β-galactosidase, producing colorless colonies in the presence of a chromogenic substrate for identifying recombinants.

4. Vectors for Plant and Animal Cloning:

   • Lessons from bacterial and viral gene delivery to eukaryotic cells have led to the development of vectors for cloning genes in plants and animals.
   • Examples include Agrobacterium tumifaciens’ Ti plasmid and modified retroviruses, which can deliver genes of interest into host cells without causing pathogenic effects.

9.2.3 Competent Host (For Transformation with Recombinant DNA) 

1. Competent Hosts for Transformation:

   • DNA is hydrophilic and cannot pass through cell membranes due to its large size and charge.
   • Bacterial cells are made “competent” for DNA uptake by treating them with a divalent cation like calcium. This treatment increases the efficiency of DNA entry through pores in the cell wall.

2. Transformation Process:

   • Bacterial cells are incubated with recombinant DNA on ice to allow binding.
   • Cells undergo a brief heat shock at around 42°C, which facilitates DNA uptake.
   • Placing the cells back on ice helps stabilize them and enhances DNA uptake efficiency.

3. Alternative Methods for DNA Introduction:

   • Micro-injection: Direct injection of recombinant DNA into the nucleus of animal cells.
   • Biolistics or Gene Gun: Bombarding plant cells with high-velocity micro-particles (gold or tungsten) coated with DNA to introduce DNA into the cells.
   • Disarmed Pathogen Vectors: Using modified pathogens (e.g., viruses or bacteria) to transfer recombinant DNA into host cells. The pathogen vectors have been rendered non-pathogenic but retain their ability to transfer DNA.