BIOTECHNOLOGY

PRINCIPLES AND PROCESSES

 BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
Biotechnology term given by :- Karl Eraky
BIOTECHNOLOGY – It is use of organisms or their
component to produce products which are useful for
humans.

ERAS OF BIOTECHNOLOGY

OLD/TRADITIONAL NEW/MODERN
 BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
Old/Traditional Biotechnology :-
Use of natural capabilities of micro organisms
to produce products .
For eg.
1. Curd making by Lactobacillus sp.
2. Wine ,Bread production by S. cerevisiae
 BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
New/Modern Biotechnology :-
Use of genetic engineering / Recombinant DNA
technology to produce products
For eg. Human insulin gene transferred in E.coli
 BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
 The European Federation of Biotechnology (EFB) has given
a definition of biotechnology that encompasses both
traditional view and modern molecular biotechnology.
 The definition given by EFB is as follows:
“The integration of natural science and organisms, cells,
parts thereof, and molecular analogues for products and
services”.
 BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
Paul Berg (Father of Genetic Engineering) :
He transferred gene of SV-40 virus into E.coli by
 – bacteriophage (Nobel prize -1980).

Stanley Cohen

Stanley Cohen and Herbert Boyer :

First Recombinant DNA produced by linking
an antibiotic resistance gene with native
plasmid of Salmonella typhimurium.
Herbert Boyer
 STEPS OF BIOTECHNOLOGY
PRINCIPLES OF BIOTECHNOLOGY :-
(1) Genetic engineering :-
To produce GMO(Genetic Modified Organism).
This Techniques alter the chemistry of genetic
material (DNA and RNA).
Modified DNA (r- DNA ) introduce into host
organisms and thus change the phenotype of the Genetic Modified
host organism. Organism(GMO)
(2) Growth of GMO at large scale level :-
Maintenance of GMO in sterile (microbial
contamination-free) environment and growth it at
large scale level for the manufacture of
biotechnological products like antibiotics, vaccines,
enzymes, etc.
TOOLS OF BIOTECHNOLOGY
Tools of Recombinant DNA Technology

Enzymes Vector or Vehicle DNA Desired gene Host cells

Lytic enzymes

Cleaving/Restriction Enzymes

Joining enzyme- Ligase

Synthesizing enzymes-
Polymerases
 TOOLS OF BIOTECHNOLOGY (ENZYME)
(I) Enzymes

(1) Lytic enzymes

They are cell degrading enzymes which
are used for isolation of DNA.
a) Lysozyme :- Digests bacterial cell wall.
b) Cellulase :- Digests plant cell wall.
c) Pectinase :- Digests plant cell wall.
d) Chitinase :- Digests fungal cell wall
e) Lipase :- Digests cell membrane
 TOOLS OF BIOTECHNOLOGY (ENZYME)
(2) Cleaving Enzymes
These enzymes are used for breaking of Nucleic acid.
They are called as Nucleases.
Cleaving enzymes are of two types :-

Exonucleases Endonucleases
Remove nucleotides from Cuts at specific positions
the ends of the DNA. within the DNA.

Eg. Restriction Endonucleases (RE)

 TOOLS OF BIOTECHNOLOGY (RE)
Restriction Endonucleases (RE) /Molecular scissor :-
1.Restriction endonuclease cuts the
phosphodiester bond (sugar – phosphate
backbone) of both strand of DNA .

2.It cut the DNA by the process of hydrolysis.

3.They cut at a specific site which is known as

Restriction site or Recognition site.

4.Restriction sites have 4/6/8 are base pairs

with palindromic nucleotide sequences.
 TOOLS OF BIOTECHNOLOGY (RE)
Palindromic Sequence :-
Sequence of nucleotides same from both ends

5' —— G A A T T C —— 3‘
3' —— C T T A A G —— 5’
 TOOLS OF BIOTECHNOLOGY (RE)
How RE cut the DNA -
a) Firstly each restriction endonuclease functions by
‘inspecting’ the length of a DNA sequence.
b) Once it finds its specific recognition sequence, it will
bind to the DNA.
c) Now RE cut between the same two bases on the
opposite strands(cut sugar -phosphate backbones).
 TOOLS OF BIOTECHNOLOGY (RE)
 RE obtain from bacteria.
 RE part of defence system of bacteria.
 It provide defence against viruses.
 RE never cut its own bacterial DNA because their
Restriction site modified by methylation.
 It never synthesized in eukaryotes.
 Till today more than 900 restriction enzymes
that have been isolated from over 230 strains of
bacteria
 TOOLS OF BIOTECHNOLOGY (RE)
Mode of cutting
(A) Oblique cut :-
 In this cut RE cut at little away from the
centre of palindromic sequence.
 It produce Sticky ends.
 e.g. :- Eco R I

GAATTC G AATTC

CTT AAG CTTAA

+ G

Sticky end
(Overhangs)
 TOOLS OF BIOTECHNOLOGY (RE)
Mode of cutting
(B) Straight cut :-
 In this Cut RE cut at the centre of palindromic sequence.
 It produce blunt ends.
 e.g. Sma I

CCCGGG CCC GGG

GGGCCC GGG
+ CCC

Blunt end
 TOOLS OF BIOTECHNOLOGY (RE)
Nomenclature Of Restriction Endonuclease-
It consist of 3/4 letter + one Roman number.
The first letter :- Indicates genus of bacteria.
Second and third letter : Indicates species of bacteria.
Fourth letter : Indicates strain of bacteria (optional).
Roman numerical : Order in which the enzymes were
isolated from bacteria.
For eg. E co R I

Genus Species Strain First enzyme isolated

(Escherichia) (coli) (RY 13) from bacteria
TOOLS OF BIOTECHNOLOGY (RE)
Recognition Sticky /
Name
sequence Blunt
1 GAATTC Sticky
Eco R I CT TAAG
2 AAG CT T Sticky
Hind III T T CGAA
Bam HI GGATCC
3 C CTAGG Sticky
GGCC Blunt
4 Hae III C CGG
5 Sma I CCC GGG Blunt
GGG CCC
6 Eco R V GATATC Blunt
CT ATAG
 TOOLS OF BIOTECHNOLOGY (ENZYME)
(3) JOINING ENZYME -DNA LIGASE (Molecular Glue)
DNA ligase join the fragments of DNA.

Gap
 TOOLS OF BIOTECHNOLOGY (ENZYME)
(4) Synthesizing enzymes
a. DNA Polymerase :- Synthesizes DNA from DNA .

DNA DNA
b. RNA Polymerase :- Synthesizes RNA from DNA .
DNA RNA

c. Reverse Transcriptase :- Synthesizes DNA from RNA.

RNA DNA
 TOOLS OF BIOTECHNOLOGY (ENZYME)
(5) Alkaline phosphatase
This enzyme remove phosphate group from 5’
end of DNA by this prevent recircularisation of
plasmid.
 TOOLS OF BIOTECHNOLOGY (VECTOR)
(II) VECTOR
Vectors are the carriers or vehicles DNA
which transfer desired gene in to the host
cell.
Features required into a Cloning Vector :-
1. Origin of replication
2. Selectable marker
3. Cloning / Restriction sites
 TOOLS OF BIOTECHNOLOGY (VECTOR)
(1) Origin of replication ( ori ) site :-
 This site Increase the number of
desired gene in to the host cell
(cloning of the gene) .

 Any piece of DNA when linked to this

sequence (Ori) can be made to
replicate within the host cells.
 TOOLS OF BIOTECHNOLOGY (VECTOR)
(1) Origin of replication ( ori ) site :-
 Ori site is also responsible for increase
and controlling the copy number of
plasmid with in the cell.
 Some plasmids may have only one or
two copies per cell whereas others
may have 15-100 copies per cell. Their
numbers can go even higher.
 TOOLS OF BIOTECHNOLOGY (VECTOR)
(2) Selectable marker gene :-
 This gene is require for selection of (NT)
transformant with recombinant cell.
 Example of selectable markers gene
(T - NR)
(a) genes encoding resistance to antibiotics
such as :-
ampicillin resistance gene (ampR), (T - R)
chloramphenicol resistance gene (chlR),
tetracycline resistance gene (terR) ,
kanamycin resistance gene (kanR).
The normal E. coli cells do not carry
resistance against any of these antibiotics
(b) Lac Z genes
 TOOLS OF BIOTECHNOLOGY (VECTOR)
(3) Cloning / Restriction sites :- Restriction site/
Cloning site
 Site of vector where desired
Eco R I
gene is to be inserted.
 Cloning site actually a Cut by Cut by
Eco R I Eco R I
restriction site where RE cut
the vector.
 TOOLS OF BIOTECHNOLOGY (VECTOR)
(3) Cloning / Restriction sites :-
 A vector should have restriction site for
many restriction enzymes, but only one
restriction site for each enzyme .
 Presence of more than one recognition
sites of one RE within the vector will
generate several fragments, which will
complicate the gene cloning