🌸Chapter 9
Biotechnology: Principles and Processes
(Notes)
1. Introduction to Biotechnology
Definition:
Biotechnology is the branch of science that uses living organisms,
cells, or biological systems to develop useful products, technologies,
and services for human welfare.
Example:
- Production of insulin, antibiotics,
vaccines, biofertilizers, GM crops, etc.
Modern biotechnology mainly involves genetic engineering and bioprocess
engineering.
2. Principles of Biotechnology
The two core principles of modern biotechnology are:
(i) Genetic Engineering
It is the technique of directly altering or manipulating
the DNA of an organism to change its phenotype.
🔹 It allows:
- Introduction of new genes
- Removal or silencing of
existing genes
- Formation of recombinant DNA
(rDNA)
Example:
Inserting human insulin gene into bacteria (E. coli) to produce
insulin.
(ii) Maintenance of Sterile
(Aseptic) Conditions
Involves maintaining microbe-free environment during
growth of desired organism.
🔹 Ensures:
- Pure culture (no contamination)
- Proper growth of genetically
modified organism (GMO)
Used in:
- Fermenters and bioreactors
3. Tools of Recombinant DNA
Technology
To perform genetic engineering, several tools are
required:
A. Restriction Enzymes (Molecular
Scissors)
Definition:
Enzymes that cut DNA at specific sequences called recognition
sites.
Discovered by Werner Arber, Hamilton Smith, and Daniel
Nathans (1978 Nobel Prize).
Types of Restriction Enzymes:
1.
Exonucleases – cut from ends of DNA.
2.
Endonucleases – cut at specific points within
DNA. (Used in rDNA technology)
Example:
EcoRI
- Recognition sequence: 5′ –
GAATTC – 3′
- Cuts between G and A → creates sticky
ends
🧠Sticky ends: Overhanging single-stranded
ends that easily join with complementary sequences.
B. DNA Ligase (Molecular Glue)
Enzyme that joins two DNA fragments by forming phosphodiester
bonds between sugar and phosphate groups.
Used to join:
- Foreign DNA + Vector DNA → Recombinant
DNA
C. Vectors
DNA molecules that carry foreign genes into a host
cell for replication and expression.
Ideal properties:
- Self-replicating
- Small in size
- Contain origin of
replication (Ori)
- Selectable marker (antibiotic resistance)
- Cloning sites (restriction
sites)
Common vectors:
1.
Plasmids – Circular DNA in bacteria (e.g.,
pBR322, pUC18)
2.
Bacteriophages – Viruses that infect bacteria
(e.g., λ phage)
3.
Cosmids,
BACs, YACs – For
larger DNA fragments
D. Host Organism
The organism in which recombinant DNA is introduced and
expressed.
Common host: E. coli
Properties:
- Rapid growth
- Easy to handle
- Known genetics
4. Processes of Recombinant DNA
Technology
Let’s understand the step-by-step process 👇
Step 1: Isolation of Genetic
Material (DNA)
Objective: Obtain pure DNA from cells.
Process:
1.
Break
open the cells → lysis using enzymes (lysozyme for bacteria, cellulase
for plant cells).
2.
Remove
RNA using RNase, and proteins using protease.
3.
Precipitate
DNA using chilled ethanol.
💡 DNA appears as fine threads.
Step 2: Cutting of DNA at Specific
Locations
- Use restriction
endonucleases to cut both vector DNA and foreign DNA at specific
sites.
- Creates sticky ends that
can pair with each other.
Step 3: Amplification of Gene of Interest
using PCR (Polymerase Chain Reaction)
Definition:
Technique to make multiple copies of a gene in vitro.
Developed by: Kary Mullis (1985)
Steps:
1.
Denaturation – DNA strands separate at 95°C
2.
Annealing – Primers attach to the template at
50–60°C
3.
Extension – DNA polymerase (Taq polymerase
from Thermus aquaticus) extends primers at 72°C
🧠Result: Millions of copies of DNA in few
hours.
Step 4: Ligation of DNA Fragments
- Join foreign DNA and vector DNA
using DNA ligase.
- Forms recombinant DNA
(rDNA).
Step 5: Insertion of Recombinant DNA
into Host (Transformation)
Methods to introduce rDNA into host:
1.
Chemical
method – CaCl₂
treatment makes bacteria competent to take up DNA.
2.
Microinjection – Directly inject DNA into the
nucleus of animal cells.
3.
Gene
gun (biolistics)
– DNA coated on gold/tungsten particles is shot into plant cells.
4.
Electroporation – Electric pulse makes cell
membrane permeable.
Step 6: Selection and Screening of
Recombinants
To identify which cells received the rDNA.
Selectable markers (antibiotic resistance genes) help in differentiation.
Example:
- Plasmid pBR322 has genes for ampicillin
and tetracycline resistance.
- Recombinants lose one
resistance → identified by antibiotic sensitivity test.
Step 7: Culturing the Recombinant
Cells
Selected cells are grown in fermenters/bioreactors
under controlled conditions:
- Temperature
- pH
- Oxygen supply
- Nutrient medium
Recombinant protein or product is then harvested and
purified.
5. Bioreactors – Large Scale
Production
Definition:
Bioreactors are large vessels that provide optimum
conditions for growth of organisms and production of desired bioproducts.
Features:
- Agitator for mixing
- Temperature and pH control
- Foam control
- Sampling ports
- Oxygen supply system
Types:
1.
Stirred-tank
bioreactor
(mechanical stirrer)
2.
Sparged-tank
bioreactor (air
bubbles mix culture)
6. Downstream Processing
After production, product undergoes purification and
packaging.
Steps:
1.
Separation of biomass
2.
Purification by chromatography, filtration, etc.
3.
Formulation (adding preservatives)
4.
Packaging
and Quality control
7. Applications of Recombinant DNA
Technology
|
Field |
Application Example |
|
Medicine |
Insulin, Growth hormone, Vaccines |
|
Agriculture |
Bt cotton, pest-resistant crops |
|
Industry |
Enzyme production, detergents |
|
Research |
Gene therapy, DNA fingerprinting |
8. Diagram Summary (Must Revise for
Exam)
- Structure of EcoRI
action site
- Steps of recombinant DNA
technology (Flowchart)
- pBR322 plasmid map
- PCR cycle diagram
- Stirred-tank bioreactor
(Labelled)
9. Important NCERT Key Terms
|
Term |
Meaning |
|
Recombinant DNA |
DNA formed by joining DNA from two sources |
|
Vector |
Carrier DNA molecule |
|
Host |
Organism receiving rDNA |
|
Restriction enzyme |
Cuts DNA at specific sequences |
|
Ligase |
Joins DNA fragments |
|
Competent cell |
Cell capable of taking foreign DNA |
|
Bioreactor |
Large vessel for product formation |
10. NEET & Board Tips
🔹 For Board:
- Revise all diagrams and
definitions from NCERT.
- Practice flowchart-based
questions.
- Remember examples of enzymes,
vectors, and processes.
🔹 For NEET:
- Focus on principles and logic —
not just names.
- Memorize restriction enzyme
recognition sequences.
- Understand PCR steps and
temperature cycle.
- Practice MCQs from NCERT lines
(direct questions come from there).
🧬 Summary Flowchart
Isolation of DNA
↓
Cutting by Restriction Enzymes
↓
Amplification (PCR)
↓
Ligation with Vector DNA
↓
Insertion into Host Cell
↓
Selection of Recombinants
↓
Culture in Bioreactor
↓
Downstream Processing → Final
Product
✅ Quick Revision Points:
- Principle: Genetic engineering
+ sterile technique
- Key enzymes: Restriction
endonuclease, DNA ligase, polymerase
- Vector: pBR322
- Host: E. coli
- Technique for gene
amplification: PCR
- Production unit: Bioreactor
- Final step: Downstream
processing
