BIOTECHNOLOGY

PRINCIPLES & PROCESSES

 Definition given by EFB ( European
Federation of Biotechnology)

“ Biotechnology is the integration

of natural science & organisms ,
cells, parts thereof, and molecular
analogues for products &
services”.
 BIOTECHNOLOGICAL PRODUCTS
&
PROCESSES.
Recombinant DNA technology.
Synthesis of a gene & introduction of
it into a target cell/organism.
Gene therapy.
In vitro fertilization for production of
test tube babies.
Biological compounds.
PRINCIPLE OF BIOTECHNOLOGY

All living organisms are composed of cells

that contain a substance called DNA. The
structure of DNA molecules contains
information that is used by cells as a
"recipe" for the organism. DNA from similar
organism is chemically and physically
similar. DNA will function if it is transferred
into any other organism.

The first recombinant DNA was constructed

by Stanley Cohen & Herbert Boyer in 1972.
STEPS IN CREATING A GMO.

The major three steps are:

1.Identification of DNA with desired
genes.
2.Introduction of the identified DNA into
a target or host cell.
3.Maintenance of introduced DNA in the
host & transfer of the DNA to its
progeny.
 TOOLS OF RECOMBINANT DNA
TECHNOLOGY.
1.Cell culture with desired DNA.
2.Restriction enzymes.
3.DNA polymerase.
4.Ligases.
5.Vector.
6.Host organism/ Cell.
RESTRICTION ENZYMES
Restriction enzymes belong to a class of
enzymes called nucleases.

Stewart Linn & Werner Arber in 1963 isolated

the first restriction endonuclease in E.coli.

H.O Smith,K.W Wilcox & T.J Kelley in 1968

isolated restriction endonuclease from
Haemophilus influenzae.
 NAMING OF RESTRICTION
ENZYMES
*The first letter of the name comes from the genus
& the next two letters from the species.
*The next letter comes from the strain.
*The roman numbers indicate the order in which
the enzymes were isolated .
EcoR I is isolated from Escherichia coli.
Hind II from Haemophilus influenzae.
Bam I from Bacillus amyloliquefaciens.
Sal I from Streptomyces albus.
Pst I from Providencia stuartii.
 Mechanism of Action of
Endonucleases
*Step-1
The restriction endonuclease inspects the entire
length of the DNA sequence.
*Step-2
The enzyme binds to the DNA at the recognition site.
*Step-3
It cuts the two strands of the double helix.
*Step-4
Sticky ends are produced as a result of cuts made.
*Step-5
The sticky ends are ligated by DNA Ligases.
Seperation & Isolation Of DNA fragments
( GEL ELECTROPHORESIS)
 Cloning Vectors

Vectors are DNA molecules that carry a

foreign DNA segment into the host cell.

Vectors may be:

1. Plasmids : A plasmid is a small,
extrachromosomal DNA molecule within
a cell that is physically separated from
chromosomal DNA and can replicate
independently.
2. Bacteriophages: These are viruses
infecting bacteria.
pBR322
Created in 1977 in the laboratory of Herbert
Boyer at the University of California, San
Francisco
It was named after the researchers who
constructed it.
The p stands for "plasmid," and BR for
"Bolivar" and "Rodriguez
They named the plasmid with the number
“322” to distinguish this vector from other
vectors they developed in their laboratory.
The following features are required to facilitate
cloning in a vector.
1.Origin of replication (Ori)
This is the sequence of base pairs on DNA where
replication starts.
2.Selectable marker
A marker is the gene which helps in selecting
those which are transformants
3.Cloning site
Vectors require few recognition sites for the
restriction enzymes
4.Small size of vector
Steps in recombinant DNA
technology.
Isolation of DNA.
Fragmentation of DNA by restriction nucleases.
Isolation of the desired DNA( Base Sequence)
fragment.
Amplification of the gene of interest by PCR
Ligation of DNA fragment into a vector
Transfer of recombinant DNA into the host
Culturing of host cells on a suitable medium
Extraction of the desired product.
Downstream processing.
Bioreactors
An apparatus for growing host organisms (yeast,
bacteria, or animal cells) under controlled
conditions.
Used in industrial processes to produce
pharmaceuticals, vaccines, or antibodies
Also used to convert raw materials into useful
byproducts such as in the bioconversion of corn
into ethanol.
Bioreactors provide optimum growth conditions
& facilitates achieving the desired product.
Components of bioreactors.

A bioreactor has the following

components:
(a) An agitator system.
(b) An oxygen delivery system.
(c) A foam control system.
(d) A temperature control system.
(e) pH control system.
(f) Sampling ports.
Bioreactor - Vessel

Head Plate

Vessel
Bioreactor - Sparger
Ring Sparger

Openings on Ring Sparger

Micro Sparger
Types of bioreactors.

1.Simple stirred tank bioreactor.

2.Sparged stirred tank bioreactor.
They are cylindrical vessels with stirrer
which facilitates the mixing & oxygen
availability throughout the bioreactor.
The difference is in the sparged stirred
tank bioreactor sterile bubbles are
sparged.
Bioreactor - Ports
Ports