Class 12 Biology Notes Chapter 11 (Biotechnology : principles and processes) – Biology Book

Alright students, let's focus on Chapter 11: Biotechnology - Principles and Processes. This is a fundamental chapter for understanding how we manipulate genetic material for human benefit, and it's crucial for various competitive exams. Pay close attention to the definitions, tools, and steps involved.

Chapter 11: Biotechnology - Principles and Processes

1. Definition of Biotechnology:

• Traditional View: Using live organisms or their enzymes to produce products and processes useful to humans (e.g., making curd, bread, wine).
• Modern View (as per EFB - European Federation of Biotechnology): The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.
• Core Techniques of Modern Biotechnology:
  • Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA), introduce these into host organisms, and thus change the phenotype of the host organism.
  • Bioprocess Engineering: Maintenance of sterile (microbial contamination-free) conditions in chemical engineering processes to enable the growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.

2. Principles of Biotechnology:

• Genetic Engineering:
  • Creates Recombinant DNA (rDNA) by combining DNA from different sources.
  • Involves Gene Cloning: Making multiple identical copies of a specific DNA segment.
  • Requires Gene Transfer: Moving the desired gene into a host organism.
  • The process relies on the concept that a piece of DNA, when transferred into an alien organism, will usually not replicate unless integrated into the host's chromosome or present as part of a chromosome segment with its own origin of replication (ori).
• Aseptic Techniques (Bioprocess Engineering):
  • Essential to prevent contamination during manipulation and large-scale culture.
  • Allows the growth of only the desired organism, leading to pure product formation.

3. Tools of Recombinant DNA Technology:

These are the essential requirements to achieve genetic engineering:

• (i) Restriction Enzymes (Molecular Scissors):

  • Enzymes that cut DNA at specific recognition nucleotide sequences.
  • Naturally found in bacteria as a defense mechanism against bacteriophages (viruses infecting bacteria). They restrict the growth of the phage. Bacteria protect their own DNA by methylation.
  • Recognition Sequence: Specific base sequence (usually 4-8 bp) recognized by the restriction enzyme. These are often palindromic sequences (read the same forwards and backwards on opposite strands, e.g., 5'-GAATTC-3' on one strand and 3'-CTTAAG-5' on the other).
  • Naming Convention:
    • First letter: Genus of the bacterium (e.g., E for Escherichia).
    • Second two letters: Species of the bacterium (e.g., co for coli).
    • Fourth letter (optional): Strain (e.g., R for R strain).
    • Roman numeral: Order of discovery from that bacterium (e.g., I for the first).
    • Example: EcoRI comes from Escherichia coli R strain, the first identified.
  • Types of Cuts:
    • Sticky Ends: Staggered cuts leaving short, single-stranded overhangs. These are useful because complementary sticky ends can base-pair (anneal).
    • Blunt Ends: Cut straight across the DNA duplex.
  • Action: Inspects DNA sequence, binds to recognition site, cuts each strand at specific points in the sugar-phosphate backbone.

• (ii) Cloning Vectors (Vehicle DNA):

  • DNA molecules that can carry a foreign DNA segment and replicate inside the host cell.
  • Used to deliver the desired gene into the host.
  • Examples: Plasmids, Bacteriophages, Cosmids, BACs (Bacterial Artificial Chromosomes), YACs (Yeast Artificial Chromosomes).
  • Essential Features of a Cloning Vector:
    • Origin of Replication (ori): Sequence where replication starts. Any DNA linked to this sequence can replicate within the host. Also controls the copy number of the linked DNA.
    • Selectable Marker: Helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants. Genes encoding resistance to antibiotics (e.g., ampicillin resistance ampR, tetracycline resistance tetR) are common selectable markers. Can also be genes like lacZ (encoding β-galactosidase).
    • Cloning Sites (Recognition Sites): Specific recognition sequences for common restriction enzymes. Ideally, a vector should have single recognition sites for several restriction enzymes, often located within one of the selectable marker genes. Ligation of foreign DNA disrupts the marker gene (insertional inactivation).
  • Example: pBR322: A common E. coli cloning vector. Contains ori, ampR gene, tetR gene, rop gene (codes for proteins involved in plasmid replication), and several unique restriction sites within the marker genes (e.g., PstI, PvuI in ampR; BamHI, SalI in tetR).
  • Insertional Inactivation: If a foreign gene is inserted into the tetR gene using BamHI, the plasmid loses tetracycline resistance but retains ampicillin resistance. Recombinants can be selected by plating on ampicillin medium (all transformants grow) and then replica plating onto tetracycline medium (non-recombinants grow, recombinants die).
  • Alternative Selection (Blue-White Screening): Based on insertional inactivation of the lacZ gene encoding β-galactosidase. If foreign DNA is inserted into lacZ, the enzyme is not produced. When grown on a chromogenic substrate (X-gal), non-recombinant colonies produce the enzyme, hydrolyze X-gal, and turn blue. Recombinant colonies (with inactivated lacZ) do not produce the enzyme and remain white.

• (iii) Competent Host (For Transformation with Recombinant DNA):

  • Host cells (usually bacteria like E. coli, yeast, or plant/animal cells) capable of taking up foreign DNA.
  • DNA is hydrophilic and cannot easily pass through the hydrophobic cell membrane.
  • Methods to make cells competent:
    • Chemical Treatment (e.g., Calcium Chloride): Treating cells with divalent cations (like Ca²⁺) increases the efficiency of DNA uptake through pores in the cell wall. This is followed by incubation on ice, a brief heat shock (e.g., 42°C), and then placing back on ice.
    • Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell using a micropipette.
    • Biolistics or Gene Gun: Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. Suitable for plant cells.
    • Disarmed Pathogen Vectors: Using modified viruses or bacteria (pathogenicity removed) to deliver DNA into host cells.

• (iv) Other Enzymes:

  • DNA Ligase: Joins DNA fragments by forming phosphodiester bonds (acts like molecular glue). Requires ATP. Used to join the foreign DNA insert to the vector DNA after both have been cut with the same restriction enzyme (generating complementary sticky ends).
  • Alkaline Phosphatase: Removes phosphate groups from the 5' ends of DNA, preventing self-ligation of the vector.
  • Reverse Transcriptase: Synthesizes complementary DNA (cDNA) from an RNA template.

4. Processes of Recombinant DNA Technology:

• (i) Isolation of the Genetic Material (DNA):

  • Break open the cell to release DNA and other macromolecules (RNA, proteins, polysaccharides, lipids).
  • Use enzymes: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi).
  • Remove RNA using Ribonuclease (RNase).
  • Remove proteins using Protease.
  • Purified DNA precipitates out after adding chilled ethanol. Seen as fine threads in suspension.

• (ii) Cutting of DNA at Specific Locations:

  • Purified DNA and vector DNA are incubated with the same specific restriction enzyme under optimal conditions.

• (iii) Amplification of Gene of Interest using PCR (Polymerase Chain Reaction):

  • Making multiple copies (billions) of a specific DNA sequence in vitro.
  • Requires: DNA template, two primers (small, chemically synthesized oligonucleotides complementary to regions flanking the target DNA), Taq polymerase (a thermostable DNA polymerase isolated from Thermus aquaticus bacterium), and deoxynucleotides (dNTPs).
  • Steps in each cycle:
    1. Denaturation: Heating (e.g., 94-96°C) to separate the two strands of the target DNA.
    2. Annealing: Cooling (e.g., 50-65°C) to allow primers to base-pair (anneal) to their complementary sequences on the separated strands.
    3. Extension: Raising temperature (e.g., 72°C, optimal for Taq polymerase) to allow the polymerase to synthesize new DNA strands starting from the primers, using the template strands.
  • These steps are repeated many times (20-30 cycles) in a thermocycler machine, leading to exponential amplification of the target DNA segment.

• (iv) Ligation of DNA Fragment into a Vector:

  • The gene of interest (cut with restriction enzyme) and the vector DNA (cut with the same enzyme) are mixed.
  • DNA Ligase is added to join the DNA fragment into the vector by forming phosphodiester bonds between the sugar-phosphate backbones. This creates recombinant DNA (rDNA) or a recombinant plasmid.

• (v) Insertion of Recombinant DNA into the Host Cell/Organism:

  • Introducing the rDNA into a competent host cell (e.g., E. coli) using methods like heat shock, electroporation, gene gun, etc. This process is called Transformation.

• (vi) Culturing the Host Cells in a Medium at Large Scale:

  • Transformed host cells are grown in suitable nutrient media under controlled conditions (temperature, pH, etc.).
  • Cells multiply and express the foreign gene, producing the desired protein (recombinant protein).
  • For large-scale production, Bioreactors are used. These are large vessels (100-1000 litres) where raw materials are biologically converted into specific products.
  • Bioreactors provide optimal growth conditions: Temperature, pH, substrate, salts, vitamins, oxygen.
  • Common type: Stirred-tank bioreactor (usually cylindrical, facilitates mixing and oxygen availability). Sparged stirred-tank bioreactors allow bubbling of sterile air.

• (vii) Extraction of the Desired Product (Downstream Processing):

  • The series of processes after the biosynthetic stage (culturing).
  • Includes: Separation and Purification of the product.
  • May involve various techniques like filtration, centrifugation, chromatography, etc.
  • Formulation with suitable preservatives.
  • Strict quality control testing is required, especially for pharmaceuticals.

This covers the core principles and processes. Remember the specific enzymes, the features of vectors, the steps in rDNA technology, and the purpose of each component.

Multiple Choice Questions (MCQs):

1. The specific DNA sequence recognized by EcoRI is:
   a) 5'-CTTAAG-3'
   b) 5'-GGCC-3'
   c) 5'-GAATTC-3'
   d) 5'-AGCT-3'

2. Which of the following is NOT an essential feature of a cloning vector?
   a) Origin of replication (ori)
   b) Selectable marker
   c) Restriction sites
   d) Ribosome binding site

3. The enzyme responsible for joining the DNA fragment to the vector DNA is:
   a) Restriction endonuclease
   b) DNA polymerase
   c) DNA ligase
   d) Reverse transcriptase

4. During Polymerase Chain Reaction (PCR), the step where primers bind to the template DNA strands is called:
   a) Denaturation
   b) Annealing
   c) Extension
   d) Ligation

5. Insertional inactivation is often used for:
   a) Amplifying the gene of interest
   b) Cutting DNA at specific sites
   c) Selecting recombinant colonies from non-recombinant ones
   d) Purifying the final protein product

6. The thermostable DNA polymerase commonly used in PCR is obtained from:
   a) Escherichia coli
   b) Agrobacterium tumefaciens
   c) Thermus aquaticus
   d) Saccharomyces cerevisiae

7. Which method is suitable for introducing recombinant DNA into plant cells?
   a) Micro-injection
   b) Heat shock method with CaCl2
   c) Biolistics (Gene Gun)
   d) Using disarmed retroviruses

8. In the nomenclature of restriction enzyme EcoRI, 'co' stands for:
   a) The strain of the bacterium
   b) The species name (coli)
   c) The genus name (Escherichia)
   d) The order of discovery

9. Downstream processing involves all the following EXCEPT:
   a) Separation of the product
   b) Purification of the product
   c) Large scale culturing in bioreactors
   d) Quality control testing

10. To isolate DNA from fungal cells, which enzyme would be required to break the cell wall?
    a) Lysozyme
    b) Cellulase
    c) Chitinase
    d) Protease

Answer Key:

1. c) 5'-GAATTC-3'
2. d) Ribosome binding site (While important for expression vectors, it's not listed as one of the essential features for a basic cloning vector in NCERT)
3. c) DNA ligase
4. b) Annealing
5. c) Selecting recombinant colonies from non-recombinant ones
6. c) Thermus aquaticus
7. c) Biolistics (Gene Gun)
8. b) The species name (coli)
9. c) Large scale culturing in bioreactors (This is part of the upstream/biosynthetic phase)
10. c) Chitinase

Study these notes thoroughly. Understand the 'why' behind each step and tool. Good luck with your preparation!