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Biotechnology???
● Technology utilizing biological system or living organism
● Developing or creating new products

Basis of Modern Biotechnology

➢ Genetic engineering − Introduction of foreign genetic material (DNA/RNA) into the host’s
genome and altering its phenotype
➢ Aseptic techniques − Involves chemical engineering processes manufacturing
products(antibiotics, vaccines) - bioprocess.

"any technological application that uses biological systems, living organisms, or derivatives
thereof, to make or modify products or processes for specific use".
Overview of biotech process
Isolation of pure DNA

Cutting of DNA Endonuclease

Separation and isolation of DNA fragment

Elution of DNA fragment Gel electrophoresis

Introduction of desired DNA fragment into vector
Plasmid-PBR322
Transfer of recombinant in to competent host
Microinjection/Biolistic
Selection of recombinants

Colonies of recombinants

Obtaining gene product Gene cloning

Bioreactors

Downstream processing
I. Isolation of the genetic material (DNA)

Bacterial cell Lysozymes

Cell wall Plant cell Cellulose

Fungus Chitinase

Plasma
membrane Lipases

RNA PROTEINS

Cytoplasm
RNAse Proteases

Precipitation - Addition of chilled

ethanol

SPOOLING-DNA-fine threads in suspension

II. Cutting of DNA at specific locations
➢ Restriction enzyme digestion - incubating purified DNA molecules

➢ 1963 - restriction enzyme isolated - they inhibit bacteriophage

growth

➢ RESTRICTION ENDONUCLEASE
○ 1st restriction endonuclease to be discovered (Hind II)
■ blunt ends or sticky ends
■ Hamilton smith & his co-workers
■ Cuts DNA - specific sequence (6bp)
■ Recognition sequence

➢ >900 restriction enzymes isolated - 230 bacterial strains

Restriction enzyme

● Prokaryotes
● Natural defense mechanism
● Prevents Bacteriophage infection

Nucleases

Exonucleases Endonucleases

Break internal
Remove
phosphodiester
nucleotides from
bond
End of DNA
Within a DNA
Special characteristics

❏ Palindromic sequence

❏ Highly specific

❏ Recognition of restriction site irrespective of DNA source

❏ Breaks phosphodiester linkage
Nomenclature
EcoRI

E co R I

Order of
Genus Species Strain identification
in bacterium

Escherichia coli RY13 First identified

III. Separation and isolation of DNA fragments
❏ By Gel electrophoresis

❏ Steps involved:
❏ Visualised using Ethidium bromide
❏ Uv light exposure-transilluminator
❏ Visible DNA bands
❏ Cutting of DNA band within gel
❏ Elution of DNA

❏ Foreign and vector DNA fragments runned under gel

electrophoresis
Recombinant DNA
Joining together of two DNA
Molecules from two different species
Foreign DNA
Vector DNA (plasmid)
IV. Formation of r-DNA

VECTOR
❏ Autonomously replicating -
cloning
❏ DNA molecule acting as
transporting vehicle
❏ Test tube to host cell
Features of a cloning vector

Replicate in host cell- using Ori

Selectable markers

Select host cell containing the vector

Unique insertional sites for cloning by RE

Minimum amount of non-essential DNA

Components of a plasmid vector
Replication start point
Ori-origin of
replication Replicate within host cell

Controls the copy number

Identify and eliminates non-transformants

Selectively permits growth of transformants

Selectable
marker Selectable markers - antibiotic resistance

Lac z coding β-galactosidase

Colour producing substrate-chromogenic

Components of a plasmid vector
Single recognition sites-to link alien DNA
More than one RS-generate several fragments

Cloning Complicate gene cloning

sites Gene of interest inserted at RE site on Antibiotic
resistant gene
Loses Antibiotic resistance

Selected out from non-recombinant ones

Size of Small as large molecule have tendency to breakdown

vector during purification
Examples of vectors used in R-DNA

1. PLASMID (e.g., PBR322)

➢ Extrachromosomal
➢ Circular
➢ Non-essential
➢ Double stranded
➢ self-replicating/autonomous
➢ Antibiotic resistance &
virulence
Nomenclature PBR322

P BR 322

Plasmid Boliver & Distinguishing

Rodriguez number
Characteristics

4.3 kb size

Two sets of Antibiotic resistant gene

High copy number

V. Amplification of gene of interest using PCR
❏ Polymerase chain reaction (PCR) - synthesis of multiple copies of
gene of interest - in vitro

❏ 2 sets of primer & enzyme DNA polymerase

❏ Primers are small-chemically synthesized OLIGONUCLEOTIDES

❏ Complementary to DNA regions

❏ Enzyme extends primers using nucleotides & genomic DNA

(template)

❏ Continuous replication-1 billion copies

VI. Insertion of recombinant DNA into the Host-cell

2. To transfer plasmid into Bacterial cell/plant cell/Animal cell

a. Heat-shock method
Transformation of plasmid DNA(foreign
plasmid/ligation product ) into E.
coli(bacteria)

SOC media(Super Optimal broth with

Catabolite repression-nutritionally rich
bacterial culture medium) is added

Transformed cells are incubated at 37

degrees C for 30 min with agitation
VI. Insertion of recombinant DNA into the Host-cell

2. To transfer plasmid into Bacterial cell/plant cell/Animal cell

b. Microinjection
Technique of delivering foreign DNA into a
living cell (a cell, egg, oocyte, embryos of
animals)

How ??? through a glass micropipette

The holding pipette holds a target cell at the

tip when gently sucked.

The tip of the micropipette is injected through

the membrane of the cell.
VI. Insertion of recombinant DNA into the Host-cell
2. To transfer plasmid into Bacterial cell/plant cell/Animal cell

c. Gene gun/biolistic

The integration of a functional fragment of DNA—DNA

construct—into target cells.

A gene construct is a DNA cassette

containing all required regulatory elements for proper

expression within the target organism.

E.g., genetically modifying plants(on seedlings or

tissue culture cell)

Prior to injecting of DNA, either microscopic gold or

tungsten particles are liberally coated with many
hundreds of copies of genes
VII. Obtaining the foreign gene product - Fermenters /
Bioreactors
➢ Chambers in which microorganism cultured in a liquid/solid medium

➢ To produce desired product in large quantities

➢ 100-1000l capacity
STIRRED TANK REACTORS
● cylindrical/curve
● Facilitate even mixing & O2 availability
● Alternative air bubbling
● Agitator
● O2 delivery system
● Foam control system
● Temperature control system
● pH control system
● Sampling ports-withdraw small cultures
Types of Bioreactors:-
VIII. Extraction of desired product

DOWNSTREAMING process

➢ Desired product recovered & purified

➢ Separation & purification of desired product

■ Filtration/centrifugation
■ drying/chromatography/solvent extraction & distillation
■ Clinical trials
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