Bio Technology

What is Biotechnology?
•Biotechnology is the use of microorganisms, plant or

animal cells or their components to generate products and
service useful to man.
•Karl Ereky coined the term biotechnology in 1919.

European Federation of Biotechnology (EFB)
“The integration of natural science and

organisms, cells, parts thereof, and molecular
analogues for products and services.”
PRINCIPLES OF BIOTECHNOLOGY
The two core techniques of modern biotechnology are:
a. Genetic engineering: b. Maintenance of sterile

ambience
The technique in which It is necessary in chemical
the genetic material engineering processes for
growing only the desired
(DNA & RNA) is microbe/ eukaryotic cell in
chemically altered and large quantities for
introduced into host the manufacture of
organisms to change the antibiotics, vaccines,
phenotype. enzymes, etc.
First recombinant DNA was

emerged from the possibility of
linking a gene of antibiotic
resistance with a native plasmid
of Salmonella typhimurium.
Stanley Cohen &Herbert Boyer

(1972) isolated the antibiotic
resistance gene by cutting out a
piece of DNA from a plasmid.
3 basic steps in genetically modifying an organism:
1. Identification of DNA with

desirable genes
2. Introduction of the
identified DNA into the host
3. Maintenance of introduced
DNA in the host and transfer of
the DNA to its progeny.
TOOLS OF RECOMBINANT DNA TECHNOLOGY
1. Restriction Enzymes (‘molecular scissors’)
These are the enzymes which cut DNA at specific
sites into fragments.
They belongs to class of enzymes called nucleases.
In 1963, two enzymes responsible for restricting

the growth of bacteriophage in E. coli were isolated.
 One of these added methyl groups to DNA.
 The other(restriction endonuclease) cut DNA.

The first restriction end nuclease is
Hind II. It always cuts DNA molecules at
a particular point by recognizing a
specific sequence of six base pairs. This
is known as the recognition sequence
for Hind II.
 Today more than 900 restriction

enzymes have been isolated from over
230 strains of bacteria.
Types of restriction enzymes
Restriction enzymes belong to a class of
enzymes called nucleases.
They include exonucleases & endonucleases.
Exonucleases
They remove nucleotides from the ends of the
DNA.
Endonucleases
They cut at specific positions within the DNA.
Each restriction endonuclease can bind to specific
recognition sequence of the DNA and cut each of
the two strands at specific points in their sugar-
phosphate backbones.
The first restriction endonuclease is Hind II.
It always cuts DNA molecules at a particular point
by recognizing specific sequence of six base pairs.
This is known as the recognition sequence for
Hind II.
Each restriction endonuclease recognizes a
specific palindromic nucleotide sequences in the
DNA.
The palindrome in DNA is a sequence of base pairs

that read the same on the two strands in 5' → 3'
direction and in 3' → 5' direction.
Restriction enzymes cut the strand a little away
from the centre of the palindrome sites, but between
the same two bases on the opposite strands. This
leaves single stranded overhanging stretches at the
ends. They are called sticky ends. They form H-bonds
with their complementary cut counterparts. This
stickiness facilitates action of the enzyme DNA ligase.
When cut by the same restriction enzyme, the

resultant DNA fragments have the same kind of
sticky-ends and these are joined together by DNA
ligases.
Separation and isolation of DNA fragments:
DNA fragments formed by restriction endonucleases can be separated by a technique
called gel electrophoresis.
The separated DNA fragments can be visualized after staining the DNA with ethidium
bromide followed by exposure to UV radiation. Bright orange coloured DNA
bands can be seen.
Separation and isolation of DNA fragments:
The separated DNA bands are cut out

from agarose gel and extracted from gel
piece. This step is called elution.
These purified DNA fragments are used

in constructing recombinant DNA by
joining them with cloning vectors.
2. Cloning Vectors
A cloning vector is a dsDNA that is used to carry the foreign gene and
replicate inside the host cell. It is also called vehicle of DNA.
E.g. Plasmids, bacteriophages , cosmids, YAC ,BAC, phagemids,
Ti –plasmid, Transposans and disarmed virus.
2. Cloning Vectors
Features of cloning vector:
2. Cloning Vectors
a.Origin of replication (ori):
 This is a sequence from where replication

starts. A piece of DNA linked to ori can
replicate within the host cells.
This also controls the copy number of the

linked DNA.
 So, for getting many copies of the target

DNA it should be cloned in a vector whose
origin support high copy number.
2. Cloning Vectors
b. Selectable marker (marker gene):

 It helps to select the transformants and
eliminate the non-transformants.
Transformation is a procedure in which a piece of
DNA is introduced in a host bacterium.
Selectable markers of E. coli include the genes
encoding resistance to antibiotics like ampicillin,
chloramphenicol,tetracycline or kanamycin, etc.
The normal E. coli cells do not carry resistance
against any of these antibiotics.
2. Cloning Vectors
c.Cloning sites:
 In order to link the alien DNA, the vector needs
very few recognition sites for restriction enzymes.
Presence of more than one recognition sites
generates several fragments, which complicates the
gene cloning.
The ligation of alien DNA is carried out at a
restriction site present in one of the two antibiotic
resistance genes.
E.g. ligation of a foreign DNA at the Bam H I site of
tetracycline resistance gene in the vector pBR322.
2. Cloning Vectors
2. Cloning Vectors
2. Cloning Vectors
2. Cloning Vectors
d. Vectors for cloning genes in plants and animals:
Genetic tools of some pathogens can be

transformed into useful vectors for delivering
genes to plants &animals.
E.g. Agrobacterioum tumifaciens

(a pathogen of many dicot plants) can deliver
a piece of DNA (T-DNA) to transform normal
plant cells into a tumor.
These tumor cells produce the chemicals

required by the pathogen.
2. Cloning Vectors
The tumor inducing (Ti) plasmid of A. Tumifaciens is modified into a cloning vector
which is not pathogenic to the plants but is able to use the mechanisms to deliver
genes of interest into plants.
Retroviruses in animals can transform normal cells into cancerous cells. So they are
used to deliver desirable genes into animal cells.
3.Competent Host (For Transformation with Recombinant DNA)
DNA is a hydrophilic molecule. So it cannot pass through cell membranes.
To avoid this problem, bacterial cells are treated with a specific concentration of
a divalent cation (e.g. calcium).
So DNA enters the bacterium through pores in cell wall.

3.Competent Host (For Transformation with Recombinant DNA)
Such cells are incubated with recombinant DNA on ice.
Then they are placed briefly at 420C (heat shock) and put them back on ice.
This enables the bacteria to take up the recombinant DNA.

Other methods to introduce alien DNA into host cells:
Micro-injection:
In this, recombinant
DNA is directly injected
into the nucleus of an
animal cell.
Biolistics (gene gun):
In this, cells are bombarded with

high velocity micro-particles of gold or
tungsten coated with DNA. This method
is suitable for plants.
‘Disarmed pathogen’ vectors:

which when infect the
cell, transfer the recombinant
DNA into the host
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
1. Isolation of the Genetic Material (DNA)
To get pure DNA (free from other macro-

molecules), the bacterial cells/plant or animal
tissue are treated with enzymes such as
lysozyme (bacteria), cellulase (plant cells),
chitinase (fungus) etc.
The cell is broken to release DNA along with

other macromolecules (RNA,proteins,
polysaccharides and lipids).
Genes (DNA) are inter wined with proteins such
as histones.
RNA is removed by treating with ribonuclease.
Proteins are removed by treatment with

protease.
Other molecules are removed by appropriate

treatments.
When chilled ethanol is added purified DNA

precipitates out as a collection of fine threads in
the suspension.
2.Cutting of DNA at Specific Locations
Restriction enzyme digestions are

performed by incubating purified DNA with
the restriction enzyme, at the optimal
conditions.
Agarose gel electrophoresis is employed to

check the progression of a restriction
enzyme digestion. As DNA is negatively
charged, it moves towards the anode.
The process is repeated with the

vector DNA also.
After cutting the source DNA and

the vector DNA, the cut out gene
(DNA segment) of interest from the
source DNA and the cut vector are
mixed and ligase is added.
This creates recombinant DNA.

3. Amplification of Gene of Interest using PCR
Polymerase Chain Reaction (PCR) is the synthesis
of multiple copies of the gene of interest in vitro
using 2sets of primers & the enzyme DNA
polymerase.
Primers are small chemically synthesized

oligonucleotides that are complementary to the
regions of DNA.
The enzyme extends the primers using the

nucleotides and the genomic DNA (template).
Through continuous DNA replication, the DNA

segment is amplified up to 1 billion copies.
4. Insertion of Recombinant DNA into the Host Cell/Organism
There are several methods of introducing the
legated DNA into recipient cells. Recipient cells
take up DNA present in its surrounding.
If a recombinant DNA bearing ampicillin resistant

gene(a selectable marker gene) is transferred into
E. coli cells, the host cells become ampicillin-
resistant cells.
If the transformed cells are spread on agar plates

containing ampicillin, only transformants will grow,
untransformed recipient cells will die.
5. Obtaining the Foreign Gene Product
 The ultimate aim of
recombinant DNA technology is to
produce a desirable protein.
The foreign gene gets expressed

under appropriate conditions.
 If a protein encoding gene is

expressed in a heterologous host,
it is called a recombinant protein.
 The cells with foreign genes may be

grown on a small scale in the laboratory.
The cultures may be used to extract the

desired protein and purify it by using
different separation techniques.
 The cells can also be multiplied in a

continuous culture system.
The used medium is drained

out from one side while fresh
medium is added from the other.
It maintains the cells more

physiologically active and so
produces a larger biomass leading
to higher yields of desired
protein.
Bioreactors
To produce large quantities of products, the bioreactors
are used where large volumes (100-1000 litres) of culture
can be processed.
Bioreactors are the vessels in which raw materials are

biologically converted into specific products, enzymes
etc., using microbial plant, animal or human cells.
A bioreactor provides the optimal growth conditions

(temperature, pH, substrate, salts, vitamins, oxygen) for
achieving the desired product.
Bioreactors
The most commonly used bioreacters are of
stirring type (stirred-tank reactor) .
It is usually cylindrical or with a curved base to
facilitate the mixing of the reactor contents.
The stirrer facilitates even mixing and oxygen
availability. Alternatively air can be bubbled
through the reactor. The bioreactor has
• An agitator system
• An oxygen delivery system
• A foam control system
• A temperature control system
• pH control system
• Sampling ports (for periodic withdrawal of the
culture)
6. Downstream Processing
 It is a series of processes such as separation and a purification of products after the

biosynthetic stage.
 The product is formulated with suitable preservatives.
Such formulation undergoes thorough clinical trials as in case of drugs.
 Strict quality control testing for each product is also required.
 The downstream processing and quality control testing vary from product to
product.

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What is Biotechnology?

•Biotechnology is the use of microorganisms, plant or

•Karl Ereky coined the term biotechnology in 1919.

“The integration of natural science and

a. Genetic engineering: b. Maintenance of sterile

First recombinant DNA was

Stanley Cohen &Herbert Boyer

1. Identification of DNA with

They belongs to class of enzymes called nucleases.

In 1963, two enzymes responsible for restricting

 One of these added methyl groups to DNA.

 The other(restriction endonuclease) cut DNA.

 Today more than 900 restriction

The palindrome in DNA is a sequence of base pairs

When cut by the same restriction enzyme, the

The separated DNA bands are cut out

These purified DNA fragments are used

 This is a sequence from where replication

This also controls the copy number of the

 So, for getting many copies of the target

b. Selectable marker (marker gene):

Genetic tools of some pathogens can be

E.g. Agrobacterioum tumifaciens

These tumor cells produce the chemicals

So DNA enters the bacterium through pores in cell wall.

This enables the bacteria to take up the recombinant DNA.

Biolistics (gene gun):

In this, cells are bombarded with

‘Disarmed pathogen’ vectors:

1. Isolation of the Genetic Material (DNA)

To get pure DNA (free from other macro-

The cell is broken to release DNA along with

Proteins are removed by treatment with

Other molecules are removed by appropriate

When chilled ethanol is added purified DNA

2.Cutting of DNA at Specific Locations

Restriction enzyme digestions are

Agarose gel electrophoresis is employed to

The process is repeated with the

After cutting the source DNA and

This creates recombinant DNA.

Primers are small chemically synthesized

The enzyme extends the primers using the

Through continuous DNA replication, the DNA

If a recombinant DNA bearing ampicillin resistant

If the transformed cells are spread on agar plates

The foreign gene gets expressed

 If a protein encoding gene is

 The cells with foreign genes may be

The cultures may be used to extract the

 The cells can also be multiplied in a

The used medium is drained

It maintains the cells more

Bioreactors are the vessels in which raw materials are

A bioreactor provides the optimal growth conditions

 It is a series of processes such as separation and a purification of products after the

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