NOTES PREPARED BY MR.

PAGADALA MALAKONDAIAH, UG NEET

BIOLOGY FACULTY.

EMAIL ADRESS: pagadalamalakondaiah143@[Link]

CONTACT NUMBER:+91-9951631887

🧬 Chapter 9: Biotechnology – Principles and Processes

Introduction

 Since the time of René Descartes (17th century), natural sciences have been directed
toward developing technologies for human comfort and welfare — a concept called
anthropocentric approach (human-centered understanding of nature).
 Physics and Chemistry led to engineering and industries; Biology mainly served as a
source of food.
 In the 20th century, biotechnology revolutionized life — improving health, food
production, and quality of life.

1. What is Biotechnology?

Traditional Sense

 Use of living organisms or enzymes from them to produce useful products/processes.

Examples:
o Making curd, bread, wine – all microbe-mediated processes.

1
Modern (Restricted) Sense

 Involves genetically modified organisms (GMOs) to perform such processes on a

large scale.

Examples of Modern Biotechnology

 In vitro fertilisation (IVF) → Test-tube baby

 Gene synthesis and its application
 DNA vaccines
 Gene therapy → Correcting defective genes

2. Definition by European Federation of Biotechnology

(EFB)
“The integration of natural science and organisms, cells, parts thereof, and molecular
analogues for products and services.”

👉 This definition includes both traditional and modern molecular biotechnology.

3. Principles of Biotechnology
Two core techniques formed the basis of modern biotechnology:

(i) Genetic Engineering

 Techniques used to alter the chemistry of genetic material (DNA/RNA).

 Allows introduction of foreign DNA into host organisms → changes their
phenotype.
 Enables transfer of desirable genes without the undesirable ones (unlike traditional
hybridisation).

(ii) Bioprocess Engineering

 Involves maintenance of sterile (contamination-free) conditions for large-scale

growth of specific microbes or eukaryotic cells.
 Ensures production of biotechnological products such as antibiotics, vaccines, and
enzymes.

2
4. Conceptual Development of Genetic Engineering
Sexual vs Asexual Reproduction

 Asexual reproduction → preserves genetic information (no variation).

 Sexual reproduction → introduces variation; sometimes beneficial.
 However, traditional hybridisation often mixes undesirable genes along with
desirable ones.

👉 Genetic engineering overcomes this by isolating and introducing only selected desirable
genes.

5. Cloning and the Role of the Origin of Replication (Ori)

 A foreign DNA transferred into another organism usually cannot replicate on its
own.
 But if it integrates into the host genome, it can replicate with the host’s DNA.
 Chromosomes contain a special sequence — origin of replication (Ori) — that
initiates replication.

👉 Therefore, any alien DNA to be cloned must be linked to a DNA segment having Ori to
allow replication.

 The process of making multiple identical copies of a DNA molecule is called cloning.

6. The First Recombinant DNA (rDNA)

 The first artificial recombinant DNA was constructed in 1972 by Stanley Cohen
and Herbert Boyer.

Steps:

1. Identification and isolation of a gene encoding antibiotic resistance from a plasmid

of Salmonella typhimurium.
2. Cutting DNA at specific sites using restriction enzymes (molecular scissors).
3. Linking the cut DNA fragment to another plasmid DNA → forms recombinant
plasmid.
4. DNA ligase enzyme joins cut DNA ends to make a stable molecule.
5. The recombinant plasmid is transferred into E. coli → the bacterium replicates the
recombinant DNA.

This demonstrated gene cloning — producing multiple copies of a specific gene using a host
organism.

7. Role of Vectors
 A vector is a carrier DNA molecule that transfers foreign DNA into a host cell.
 Plasmids act as vectors (similar to how mosquitoes transfer the malarial parasite).

3
Functions of Vectors:

 Carry foreign DNA into host cells.

 Contain origin of replication for independent replication.
 Have selectable markers (e.g., antibiotic resistance genes) to identify transformed
cells.

8. Enzymes Used in Recombinant DNA Technology

Restriction Enzymes (Endonucleases)

 Act as molecular scissors to cut DNA at specific sequences (called recognition

sites).
 Produce sticky ends that allow insertion of foreign DNA

DNA Ligase

 Acts as molecular glue – joins DNA fragments by forming phosphodiester bonds

between them.

9. Key Steps in Genetic Modification of an Organism

There are three basic steps:

1. Identification of DNA with desirable genes

– e.g., gene for antibiotic resistance or insulin production.

4
 2. Introduction of identified DNA into the host organism
– Using plasmid vectors, gene guns, microinjection, etc.
3. Maintenance and inheritance of the introduced DNA
– The introduced gene must replicate and pass on to progeny cells.

10. Contributions of Stanley Cohen and Herbert Boyer

 Herbert Boyer (b. 1936):
o Worked on restriction enzymes of E. coli.
o Discovered their ability to create sticky ends that allow DNA splicing.
 Stanley Cohen:
o Studied plasmids (small circular DNA in bacteria).
o Developed methods for plasmid removal and reintroduction.
 Their collaboration (1972) led to:
o Creation of recombinant DNA molecules.
o Birth of modern biotechnology.

5
🧩 Summary Table
Concept Description / Role
Use of microbes/enzymes to make useful products (e.g. curd,
Biotechnology (Traditional)
wine).
Use of GMOs and molecular techniques for large-scale
Modern Biotechnology
production.
Integration of natural science and organisms for
Definition (EFB)
products/services.

6
Concept Description / Role
Core Techniques Genetic engineering and bioprocess engineering.
Genetic Engineering Modification of genetic material and introduction into host.
Bioprocess Engineering Maintenance of sterile conditions for large-scale culture.
Origin of Replication (Ori) DNA sequence needed to initiate replication.
Plasmid Vector Carries foreign DNA into host; replicates independently.
Restriction Enzyme Cuts DNA at specific sequences to create sticky ends.
DNA Ligase Joins cut DNA fragments to form recombinant DNA.
Recombinant DNA Artificially combined DNA from different sources.
Cloning Making multiple identical copies of DNA.
3 Steps in Gene
Identify, Introduce, Maintain & Transfer DNA.
Modification

In Essence
Modern biotechnology stands on two pillars — genetic engineering and bioprocess
engineering.
It allows scientists to precisely manipulate genes, create recombinant DNA, and mass-
produce valuable products such as hormones, enzymes, antibiotics, and vaccines,
transforming healthcare and agriculture.

🧬 Chapter 9 – Biotechnology: Principles

and Processes

7
9.2 Tools of Recombinant DNA Technology
To perform genetic engineering (recombinant DNA technology) successfully, several key
tools are required.

🔧 The Five Essential Tools

1. Restriction enzymes – for cutting DNA at specific sites
2. Polymerase enzymes – for synthesizing new DNA/RNA strands
3. Ligases – for joining DNA fragments
4. Vectors – for carrying foreign DNA into host cells
5. Host organism – for replication, expression, and propagation of recombinant DNA

In this section, we focus mainly on restriction enzymes, gel electrophoresis, and isolation
of DNA fragments.

🧩 9.2.1 Restriction Enzymes

🧠 Historical Background

 Discovered in 1963 during studies on Escherichia coli (E. coli) and bacteriophages.
 Two types of enzymes were found:
1. One added methyl groups to DNA (methylase).
2. The other cut DNA — called restriction endonuclease.

8
  These enzymes restrict the growth of bacteriophages in bacteria — hence the name
restriction enzymes.

🔬 First Restriction Enzyme

 Hind II was the first restriction endonuclease isolated (in 1968).

 It recognizes a specific sequence of six base pairs and always cuts at the same point
within this sequence.
 The recognized sequence is called the recognition sequence or restriction site.
 For Hind II, the recognition sequence is:
 5' — GTY'RAC — 3'

(where Y = pyrimidine, R = purine)

 Today, over 900 restriction enzymes have been isolated from more than 230
bacterial strains, each recognizing a unique sequence.

🧬 Naming Convention of Restriction Enzymes

 The name of each enzyme is derived from the bacterium from which it was isolated.

Component Meaning Example: EcoRI

1st letter Genus name E – Escherichia
2nd & 3rd letters Species name co – coli
4th letter Strain R – strain RY13
Roman numeral Order of discovery I – first enzyme isolated from that strain

👉 Thus, EcoRI = Escherichia coli strain RY13, first enzyme isolated.

🔍 Classification of Nucleases

Type Function
Exonucleases Remove nucleotides from the ends of DNA.
Endonucleases Cut DNA within the molecule at specific sites.

🔹 Restriction endonucleases belong to this second category — they cut DNA at specific
recognition sequences.

9
🧫 Mechanism of Restriction Endonuclease Action

1. The enzyme scans the DNA for its specific recognition site.
2. When it finds this palindromic sequence, it binds and cuts both strands at precise
locations.
3. The cut produces short single-stranded overhangs called sticky ends (or cohesive
ends).

10
🧬 What are Palindromic Sequences?

 A palindrome is a sequence that reads the same forward and backward.

o Example (word palindrome): MALAYALAM
 In DNA, a palindromic nucleotide sequence reads the same on both strands when
read in the 5′ → 3′ direction.

Example: Recognition site of EcoRI

5′ — GAATTC — 3′
3′ — CTTAAG — 5′

👉 Reading both strands from 5′ → 3′ gives the same sequence.

🧷 Formation of Sticky Ends

 Restriction enzymes cut slightly away from the center of the palindromic site.
 This creates short overhanging single-stranded regions called sticky ends.

👉 These sticky ends can form hydrogen bonds with complementary sticky ends from other
DNA fragments cut by the same enzyme.
👉 This base pairing facilitates joining by DNA ligase, forming recombinant DNA.

🧬 Importance of Using the Same Restriction Enzyme

 The vector DNA (carrier) and foreign DNA (gene of interest) must be cut with the
same restriction enzyme.
 This ensures that both produce complementary sticky ends, enabling them to join
perfectly to form recombinant DNA.

11
🧩 Role of DNA Ligase

 After restriction digestion, DNA ligase joins the cut DNA fragments by forming
phosphodiester bonds.
 It seals the nicks between the sugar-phosphate backbones of DNA strands.
 Result: formation of a stable recombinant DNA molecule.

⚡ Separation and Isolation of DNA Fragments

After cutting DNA with restriction enzymes, we obtain multiple DNA fragments of
different lengths.
These fragments must be separated and purified before being joined into vectors.

⚙️Technique: Gel Electrophoresis

Principle:

 DNA fragments are negatively charged (due to phosphate groups).

 When placed in an electric field, they migrate toward the anode (+).
 The separation occurs through a gel matrix, which acts as a molecular sieve.

Medium Used:

12
  The commonly used matrix is agarose gel, a natural polysaccharide extracted from
seaweeds.

Process:

1. DNA sample is loaded into wells at one end of the agarose gel.
2. An electric field is applied — DNA moves toward the positive end.
3. Smaller DNA fragments move faster and farther than larger ones because of the
sieving effect.
👉 Hence, fragment size is inversely proportional to the distance migrated.

Visualization of DNA:

 DNA is invisible under normal light, so it must be stained.

 The gel is soaked in ethidium bromide, which binds to DNA.
 Under UV light, the DNA bands appear as bright orange bands.

Extraction of DNA Fragments (Elution):

 Specific bands corresponding to desired DNA fragments are cut out from the gel.
 The DNA is extracted and purified — this process is called elution.
 The purified DNA fragments are then used for constructing recombinant DNA by
joining them to cloning vectors.

13
🧠 Summary Table: Key Concepts
Tool / Process Description Key Example / Function
Cuts DNA at specific sites (restriction
Restriction enzyme EcoRI, HindII
sites)
Recognition Specific DNA sequence (palindrome)
GAATTC (EcoRI site)
sequence where enzyme cuts
Overhanging single-stranded ends after
Sticky ends Facilitate ligation
cut
DNA ligase Joins DNA fragments covalently Forms phosphodiester bonds
Vector Carrier DNA molecule Plasmid
Smaller fragments move
Gel electrophoresis Separates DNA fragments by size
farther
Agarose gel Medium for DNA separation Extracted from seaweed
Shows orange bands under
Ethidium bromide DNA stain
UV
Used for recombinant DNA
Elution Extraction of DNA from gel
formation

🧬 Flow Summary of the Process

1. DNA Cutting:
o Restriction enzyme (e.g., EcoRI) cuts vector & foreign DNA → produces
sticky ends.
2. Fragment Separation:
o Fragments separated by gel electrophoresis.
3. Visualization & Extraction:
o DNA bands visualized under UV after ethidium bromide staining → desired
fragment eluted.
4. Joining:
o Sticky ends of vector and foreign DNA joined by DNA ligase → forms
recombinant DNA molecule.

📘 Key Terms at a Glance

Term Meaning
Restriction endonuclease Enzyme cutting DNA at specific internal sites
Exonuclease Enzyme removing nucleotides from DNA ends
Recognition sequence Specific DNA sequence recognized by enzyme
Palindromic sequence Sequence that reads same in both directions
Sticky ends Short overhanging ends formed after cut
DNA ligase Enzyme sealing DNA fragments together
Agarose gel electrophoresis Technique to separate DNA fragments
Ethidium bromide Fluorescent dye used to visualize DNA
Elution Extraction of DNA from gel for further use

14
🧠 Concept Check (NEET Level Quick Recall)
1. Who discovered restriction enzymes?
→ Found in E. coli (1963); first enzyme Hind II characterized later (1968).
2. What is the recognition sequence of EcoRI?
→ 5′–GAATTC–3′
3. Why are sticky ends important?
→ Facilitate the joining of DNA fragments by complementary base pairing.
4. What determines how far DNA fragments move in electrophoresis?
→ Their size — smaller fragments move farther.
5. What is elution?
→ The process of cutting out and purifying specific DNA fragments from a gel.

🔬 9.2.2 Cloning Vectors

🧭 Meaning of a Cloning Vector

 A cloning vector is a DNA molecule that can carry a foreign DNA fragment into a
host cell and replicate there.
 The most common cloning vectors are plasmids and bacteriophages.

🧫 Why Plasmids and Bacteriophages Are Used

 Both have the ability to replicate independently of the host’s chromosomal DNA.
 Bacteriophages → have high copy numbers (many phage genomes per infected
bacterial cell).
 Plasmids → may have 1–2 copies (low-copy plasmids) or 15–100+ copies per cell
(high-copy plasmids).
 When a foreign (alien) DNA is linked to plasmid or phage DNA, it can be amplified
along with them.

15
🧩 Essential Features of a Cloning Vector

To efficiently clone and select recombinant DNA, a vector must have four main features:

(i) Origin of Replication (ori)

 A specific DNA sequence from where replication starts.

 Controls:
o Copy number (how many copies of plasmid/DNA will be formed per cell).
o Maintenance of plasmid inside the host.
 Importance:
→ When a foreign DNA is linked to the vector’s ori sequence, it gets replicated inside
the host cell.
 For high yield of target DNA → choose vectors with high-copy-number origins.

16
(ii) Selectable Marker

 Used to identify and select transformed cells (those that have taken up the vector)
from non-transformants.
 Transformation = process of introducing a vector DNA into a host bacterium.
 Selectable markers are usually antibiotic resistance genes such as:
o ampR – resistance to ampicillin
o tetR – resistance to tetracycline
o camR – resistance to chloramphenicol
o kanR – resistance to kanamycin
 Normal E. coli cells are sensitive to these antibiotics.
 So only those that received the plasmid (transformants) will survive on antibiotic
medium.

Example: pBR322 Vector

 One of the most commonly used E. coli plasmid cloning vectors.

 Contains:
o ori – origin of replication
o ampR and tetR – antibiotic resistance genes (ampicillin & tetracycline)
o Unique restriction sites for enzymes like EcoRI, BamHI, PstI, SalI, ClaI,
HindIII, PvuII
o rop gene – codes for proteins that regulate plasmid replication.

17
Selection of Recombinants Using pBR322

 Suppose the foreign DNA is inserted at BamHI site (inside tetR gene):
o This disrupts tetR → tetracycline resistance is lost.
o ampR remains functional → ampicillin resistance retained.
 Thus:
o Transformants grow on ampicillin plates.
o Recombinants (with foreign DNA) cannot grow on tetracycline.
 Screening Procedure:

1. Plate cells on medium with ampicillin → only transformants survive.

2. Replica plate on tetracycline → those that fail to grow here are recombinants
(insertional inactivation of tetR).

(iii) Cloning / Recognition Sites

 Vectors must have a few unique restriction sites (ideally only one site for each
enzyme).
 Enables insertion of foreign DNA at a single location, avoiding fragmentation of the
vector itself.
 Example: BamHI, EcoRI, HindIII in pBR322.

18
  Multiple sites would complicate cloning by producing too many fragments.

(iv) Insertional Inactivation & Colour-Based Screening

 Instead of using antibiotics, sometimes insertional inactivation of a reporter gene is

used for easier detection.
 Example: lacZ gene encoding β-galactosidase enzyme.
o If no insert → functional enzyme → blue colonies on medium with
chromogenic substrate (X-gal).
o If insert present → enzyme inactivated → white colonies.
 This Blue-White Screening simplifies identification of recombinants.

(v) Vectors for Plants and Animals

 Plants: Modified Ti plasmid (Tumour-inducing plasmid) of Agrobacterium

tumefaciens.
o Natural pathogen of dicots; transfers T-DNA to plant genome causing tumors.
o Scientists have disarmed Ti plasmid (removed disease genes) → now used as
a vector to deliver desired genes into plant cells.
 Animals: Modified retroviruses are used as vectors.
o Normally cause cancer by inserting their genome into host DNA.
o Disarmed retroviruses can deliver therapeutic or experimental genes safely
into animal cells.

✅ Summary of Features of a Good Cloning Vector

Feature Function
Origin of replication (ori) Enables replication & controls copy number
Selectable marker Identifies transformants from non-transformants
Cloning/Restriction site For insertion of foreign DNA
Reporter/Indicator gene For visual selection of recombinants
Size Small size preferred for easy manipulation
Stability Should remain stable inside host
Host range Should replicate in chosen host organism

🧬 9.2.3 Competent Host (For Transformation with

Recombinant DNA)
🧠 Concept

 DNA is hydrophilic → cannot naturally pass through the cell membrane.

 So, bacterial cells (usually E. coli) must be made competent to take up plasmid DNA.

⚗️Steps to Make Bacterial Cells Competent

1. Chemical Treatment
o Treat cells with a divalent cation (commonly CaCl₂).

19
 o This weakens the cell wall and increases permeability by creating temporary
pores.
o Positively charged Ca²⁺ ions neutralize the negative charge on DNA and cell
membrane → facilitates DNA binding.
2. Heat-Shock Treatment
o Incubate mixture of competent cells + recombinant DNA on ice.
o Expose briefly to 42°C (heat shock) → creates a thermal imbalance → DNA
enters the cell.
o Immediately return to ice to stabilize the membranes.

🔁 This process increases transformation efficiency.

🔫 Alternative Methods of Gene Transfer

Method Principle Used In

Direct injection of recombinant DNA into the Animal
Microinjection
nucleus of animal cells using a fine needle cells
High-velocity microparticles (gold/tungsten) coated
Biolistics (Gene Gun) Plant cells
with DNA are shot into target cells
Pathogens (like Agrobacterium or retrovirus) are
Disarmed Pathogen- Plants &
modified to deliver desired genes without causing
Mediated Transfer animals
disease

20
21
🧩 Transformation Summary

1. Prepare competent host cells.

2. Introduce recombinant DNA by one of the above methods.
3. Select transformants using selectable markers.
4. Allow multiplication of the foreign DNA inside the host.

🧾 Key Terms
Term Definition
Vector DNA molecule used to carry foreign DNA into host cell
Plasmid Small circular extra-chromosomal DNA in bacteria
Transformation Uptake of foreign DNA by a host cell
Insertional Inactivation Inactivation of a marker gene by insertion of foreign DNA
Selectable Marker Gene used to identify transformants
Competent Cell Cell capable of taking up foreign DNA
Biolistics DNA delivery using particle bombardment

🧠 Concept Map
Recombinant DNA Technology
↓
Requires a Vector
↓
Features of Vector
(ori, marker, cloning site, etc.)
↓
Foreign DNA inserted → Recombinant DNA
↓
Transformation into Competent Host
(CaCl₂, Heat shock, Gene gun, etc.)
↓
Selection of Recombinants
(Antibiotic/Color screening)

🧪 NEET/CBSE Quick Recall Points

1. pBR322 – first artificial cloning vector in E. coli.
2. ampR & tetR genes – act as selectable markers.
3. Insertional inactivation – loss of marker gene function due to insertion of foreign
DNA.
4. Blue-white screening – based on lacZ gene and chromogenic substrate (X-gal).
5. Ti plasmid – vector for plants (from Agrobacterium tumefaciens).
6. Retrovirus – vector for animals.
7. Competent cells – treated with CaCl₂, exposed to 42°C heat shock.
8. Biolistics – gene gun used for plant transformation.
9. Microinjection – DNA injected into animal cell nucleus.
10. Disarmed pathogens – modified to deliver genes without causing disease.

22
🌿 9.3 PROCESSES OF RECOMBINANT
DNA TECHNOLOGY
Recombinant DNA technology is a step-wise process that allows scientists to isolate a
specific gene, insert it into a suitable vector, transfer it into a host organism, and produce
large quantities of the desired product (usually a protein).

🔶 Overview of Major Steps

1. Isolation of genetic material (DNA)
2. Cutting of DNA at specific locations
3. Amplification of gene of interest using PCR
4. Insertion of recombinant DNA into host cell
5. Obtaining the foreign gene product (expression)
6. Large-scale production using bioreactors
7. Downstream processing (purification and formulation)

🧬 9.3.1 Isolation of the Genetic Material (DNA)

🔹 Purpose

To obtain pure DNA from cells, free from RNA, proteins, lipids, and polysaccharides.

🔹 Principle

DNA is enclosed within the cell membrane and complexed with proteins; therefore, the cell
must be broken open and impurities removed.

🔹 Step-by-Step Process

Step Description
The cell wall and membrane are disrupted using specific enzymes: –
1. Cell Lysis
Lysozyme → for bacteria (breaks peptidoglycan wall) – Cellulase →
(Breaking Open
for plants (digests cellulose wall) – Chitinase → for fungi (digests
Cells)
chitin wall)
2. Removal of – Protease removes proteins (including histones) – Ribonuclease
Proteins and RNA (RNase) removes RNA
3. Separation of – DNA is precipitated using chilled ethanol – DNA appears as fine,
DNA thread-like fibers (can be seen as threads spooled out with a glass rod).
4. Purification Further washing removes salts and impurities → pure DNA obtained.

🧠 Key Concept

 DNA is hydrophilic and remains in aqueous phase.

 Addition of ethanol (nonpolar solvent) dehydrates and precipitates DNA.

23
24
✂️9.3.2 Cutting of DNA at Specific Locations
🔹 Purpose

To cut DNA molecules (both vector and donor DNA) precisely at specific sequences using
restriction endonucleases.

🔹 Process

1. DNA is mixed with a specific restriction enzyme and incubated under optimal
conditions (specific temperature, pH, and buffer).
2. The enzyme cuts DNA at its recognition site (usually palindromic sequences).
3. Both vector DNA and foreign DNA are cut with the same enzyme → compatible
“sticky ends”.
4. The cut DNA fragments are visualized using agarose gel electrophoresis:

25
 o DNA is negatively charged → migrates towards anode (+).
o Fragments are separated by size (smaller fragments move faster).

🔹 Ligation

 The desired gene fragment and cut vector DNA are mixed together.
 DNA ligase joins the sugar-phosphate backbones → forming recombinant DNA.

🧩 Result:
A vector (e.g., plasmid) carrying the gene of interest is now ready for transfer into a host.

🔁 9.3.3 Amplification of Gene of Interest Using PCR

🔹 Definition

PCR (Polymerase Chain Reaction) is a technique to produce millions of copies of a DNA

segment in vitro (outside the cell).

🔹 Components Required

1. Template DNA – contains the target gene.

2. Primers – short synthetic oligonucleotides complementary to target DNA ends.
3. Taq DNA polymerase – thermostable enzyme from Thermus aquaticus.
4. dNTPs (deoxynucleotides) – building blocks for new DNA synthesis.
5. Buffer & Mg²⁺ ions – maintain optimal enzyme activity.

🔹 Three Main Steps in Each PCR Cycle

Step Description Temperature

1. Denaturation Double-stranded DNA melts into single strands. ~94–95°C

26
 Step Description Temperature
Primers bind (anneal) to complementary regions on
2. Primer Annealing ~50–60°C
DNA template.
3. Taq polymerase adds nucleotides to extend primers
~72°C
Extension/Elongation → new strand synthesis.

Each cycle doubles the DNA amount → 30 cycles ≈ 1 billion copies of target DNA.

🔹 Applications

 Amplified DNA can be:

o Cloned into a vector.
o Sequenced.
o Used for gene expression.
o Used in diagnostics (e.g., COVID, HIV, genetic tests).

🧫 9.3.4 Insertion of Recombinant DNA into the Host

Cell/Organism
🔹 Purpose

To transfer recombinant DNA into a suitable host cell (bacteria, plant, or animal) for
replication and expression.

🔹 Methods
27
 1. Chemical method → (CaCl₂ + heat shock):
o Host E. coli cells are made competent and incubated with recombinant
plasmid.
o Heat shock at 42°C allows plasmid entry.
2. Microinjection → DNA directly injected into the nucleus (animal cells).
3. Biolistics (Gene Gun) → DNA-coated gold/tungsten particles shot into plant cells.
4. Disarmed Pathogen Vectors → Agrobacterium tumefaciens (plants) or retroviruses
(animals) used to deliver DNA safely.

🔹 Selection of Transformants

 Example: If recombinant plasmid carries ampicillin resistance gene (ampR):

o Cells with recombinant plasmid grow on ampicillin plates.
o Cells without plasmid (non-transformants) die.

🧠 Selectable Marker: The ampR gene acts as a marker to identify transformed cells.

🧪 9.3.5 Obtaining the Foreign Gene Product

🔹 Goal

To express the inserted gene and produce the desired protein.

🔹 Expression

 Once inside the host, the recombinant DNA replicates and the foreign gene is
expressed using the host’s transcription and translation machinery.
 The expressed gene product (usually a protein or enzyme) is called a recombinant
protein.

🔹 Scaling Up Production

Step Description
Small
Recombinant bacteria/plant/animal cells grown in laboratory flasks.
Scale
Large Grown in bioreactors (100–1000 L). Continuous culture system maintains
Scale growth phase for high yield.

⚙️Bioreactors
🔹 Definition

Large vessels in which raw materials are biologically converted into specific products
(proteins, enzymes, etc.) using living cells or enzymes under controlled conditions.

🔹 Functions

28
Provide optimum conditions for:

 Temperature
 pH
 Oxygen concentration
 Nutrient supply
 Agitation and mixing

🔹 Types of Bioreactors

Type Description
Stirred-Tank Cylindrical vessel with curved base for easy mixing; mechanical
Bioreactor stirrer ensures uniformity.
Sparged Stirred-
Air is sparged (bubbled) through culture to improve aeration.
Tank

Components of a Bioreactor

1. Agitator system – for mixing and aeration.

2. Oxygen delivery system – maintains dissolved oxygen.
3. Foam control system – prevents overflow.
4. pH and temperature control – via sensors and regulators.
5. Sampling ports – for periodic testing of product.

🔹 Continuous Culture System

 Spent medium is removed and replaced with fresh medium continuously.

 Maintains cells in log/exponential growth phase, ensuring maximum yield.

🧴 9.3.6 Downstream Processing

🔹 Definition

29
All processes after biosynthesis of the product to make it ready for marketing.

🔹 Steps Involved

1. Separation – isolate cells or secreted product from the culture medium.

2. Purification – remove impurities using chromatographic and filtration methods.
3. Formulation – add preservatives, stabilizers, or buffers for long shelf life.
4. Quality Control & Clinical Trials – to ensure safety, efficacy, and compliance with
regulatory standards.

🔹 Importance

 Ensures purity, safety, and effectiveness of biotechnological products like insulin,

interferons, vaccines, etc.
 Downstream processing can account for 60–80% of the total production cost.

🧠 Summary Table of rDNA Technology Steps

Step Process Key Tools/Concepts
Cell lysis & enzyme
1. Isolation of DNA Lysozyme, cellulase, protease
treatment
Restriction enzymes, gel
2. Cutting of DNA Restriction digestion
electrophoresis
3. Amplification PCR Taq polymerase, primers
4. Ligation & Insertion Joining DNA fragments DNA ligase, vector
5. Transformation Transfer to host Competent cells, selectable markers
6. Expression Protein production Host transcription/translation
7. Large-scale culture Growth in reactors Bioreactors
8. Downstream
Purification & formulation Filtration, chromatography
processing

⚡ NEET & CBSE Key Points

1. Restriction enzymes create sticky ends → allow ligation.
2. Taq polymerase is thermostable → survives at 95°C.
3. ampR gene – selectable marker for transformed E. coli.
4. Bioreactor volume – 100–1000 L (sometimes more in industry).
5. Downstream processing = purification + formulation.
6. Recombinant proteins – proteins produced by foreign gene expression (e.g., insulin,
interferon).
7. Continuous culture → keeps cells in exponential phase for high yield.
8. Agarose gel electrophoresis – DNA moves to anode (+) due to negative charge.
9. Cellulase/Chitinase/Lysozyme – used for breaking plant/fungal/bacterial cell walls.
10. Ethyl alcohol used to precipitate pure DNA.

🧾 Final Summary of the Whole Process

30
 ⃣ Isolation of DNA
1️
↓
⃣ Cutting with restriction enzymes
2️
↓
⃣ Ligation with vector → Recombinant DNA
3️
↓
⃣ Transfer into host → Transformation
4️
↓
⃣ Selection of transformants
5️
↓
⃣ Expression of gene → Recombinant protein
6️
↓
⃣ Large-scale culture → Bioreactor
7️
↓
⃣ Downstream processing → Purified product
8️

31