Bio. tech. pr.& process

BIOTECHNOLOGY :
PRINCIPLES AND
PROCESSES
By:-
V.S.Malik
Biology Lecturer (Retd.)

WHAT IS BIOTECHNOLOGY?
 Biotechnology refers to the technology using biology. It has
applications in various fields like: agriculture, food processing
industry, medicine diagnostics, bioremediation, waste
treatment and energy production.
 Biotechnology deals with the techniques of using live
organisms or a component of an organism or enzymes from
organisms or other biological system, to make products and
processes that benefit human beings.
 Man has been using the biological processes of
microorganisms for more than 6,000 years to make useful
food products, such as bread and cheese, and to preserve
dairy products.”

BIOTECHNOLOGY
 According to the Biotechnology Innovation Organization,
 “Biotechnology is the technology based on biology –
biotechnology harnesses cellular and bio-molecular
processes to develop technologies and products that help
improve our lives and the health of our planet.”
 The definition of Biotechnology given by European
Federation of Biotechnology (EFB) is as follows:
“The integration of natural science and organisms, cells,
parts thereof, and molecular analogues for products and
services is called biotechnology.”

DIFFERENT FIELDS/TYPES OF BIOTECHNOLOGY
Industrial Biotechnology.
Environmental Biotechnology.
Microbial Biotechnology.
Agricultural Biotechnology.
Animal Biotechnology.
Forensic Biotechnology.
Aquatic Biotechnology.
Medical Biotechnology and Bioremediation.
Regulatory Biotechnology.

PRINCIPLES OF BIOTECHNOLOGY
 Modern biotechnology is the result of the
following two core techniques -
 Genetic Engineering : It includes several
techniques that facilitate the alteration of
genetic material, that is, RNA or DNA, in order
to introduce them in host organisms. It includes
changing of phenotype in host organism.
Aseptic techniques : In chemical engineering
process, maintenance of microbial contamination
free (sterile) atmosphere with an objective to
initiate the large growth of desired eukaryotic
or microbe cell in order to manufacture
biotechnological products like enzymes, vaccines
and antibiotics.

TECHNIQUES OF
GENETIC
ENGINEERING
•1. Construction of
artificial
recombinant DNA
•2. Gene transfer
into host organism
•3. Gene cloning

CONSTRUCTION OF RECOMBINANT DNA
 Stanley Cohen and Herbert Boyer
(1972) constructed the first
recombinant DNA.
 They isolated the antibiotic
resistance gene from the native
Plasmid of the bacterium
Salmonella typhimurium.
 This piece of DNA carrying
antibiotic resistance gene was cut
at specific location by restriction
endonuclease enzyme, popularly
called as Molecular Scissors.

GENE TRANSFER
The cut piece of DNA was
introduced in the plasmid of
Escherichia coli which acted as
the vector.
The piece of DNA was ligated to
the vector plasmid by DNA ligase.
This joining of the two DNA
pieces resulted in the creation of
recombinant DNA.
Escherichia coli

GENE CLONING
The new recombinant DNA
was transferred into E. coli.
The rDNA replicated
autonomously by using the
host DNA polymerase
enzyme and made multiple
copies.
The ability to multiply
copies of any template of
DNA is called gene cloning.

BASIC REQUIREMENTS FOR GENETIC MODIFICATIONS
Identification of DNA with
desired genes.
Introduction of desired
identified genes into host.
Maintenance of introduced
DNA in the host.
Transfer of this DNA to its
progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY
Restriction enzymes
Cloning vehicles
(Vectors)
DNA polymerase
enzyme
DNA Ligase enzyme
Competent host
organism

RESTRICTION ENZYMES
They are known as biological scissors.
Restriction enzymes belong to a larger
class of enzymes called nucleases.
They are of two types: exonucleases
and endonucleases.
Exonucleases removes the
nucleotides from the ends of DNA.
Endonucleases make cuts at specific
positions within DNA.

RESTRICTION ENZYMES
 Three main types of restriction enzymes:
 Type I, Type II and Type III. Type II are used
for molecular biology work.
 The first restriction endonuclease Type II
enzyme isolated was Hind II in 1970.
 It was isolated from the bacterium
Haemophilus influenzae.
 Today we know more than 3000
restriction enzymes isolated from 230
strains of bacteria and more than 600 of
these are available commercially.

RESTRICTION ENZYMES
 The specific base sequence where a
restriction enzyme cut the DNA molecule
is known as recognition sequence.
 The recognition sequence for Type II
restriction enzymes form pallindromes.
 Palindromes are group of letters that
form the same words when read both
forward and backward e.g. MALYALAM. It
is read the same on two strands of DNA
when orientation is kept the same.
 EcoRI recognises only the following
sequence.
5’-------GAATTC-------3’
3’-------CTTAAG-------5’

5’-ATGCGAT-3’
3’-TACGCTA-5’
Eco RV
5’-ATGCG-3’
3’-TACGCTTAA-5’
Eco R1
5’ ATGCGAATTCCGGAA 3’
3’ TACGCTTAAGGCCTT 5’
5’ ATGCGATATCCGGAA 3’
3’ TACGCTATAGGCCTT 5’
Sticky ends
Blunt ends
5’-AATTCCGGAA-3’
3’-GGCCTT-5’
5 ’-ATCCGGAA-3’
3’-TAGGCCTT-5’
Sticky and Blunt ends
Endonucleases
recognize specific
sequence of
base-pairs,
usually 4, 6 or 8
bases that are
palindromic.
They can leave
‘sticky ends’ or
‘blunt ends’

NOMENCLATURE OF RESTRICTION ENZYMES
 First letter of genus is written in capital.
 Next two letters of species in lower case.
 All the above three letters should be
written in italics, e.g. Eco from Escherichia
coli and Hin from Haemophilus inflenzae.
 This is followed by strain, e.g. Eco R.
 e.g. Eco RI comes from Escherichia coli
RY13. Where R is derived from the strain.
 Roman number indicate the order in
which enzymes were isolated from the
strain of bacteria.

RESTRICTION ENZYME ACTION OF Eco RI

Role of Restriction Enzymes
• They help in generating DNA fragments with precise
ends.
• These precisely generated ends can then be ‘pasted’ to
similar ends in a self replicating vector molecule.
• The process of ‘pasting’ is called ligation and requires
DNA ligase enzyme.
• The product after ligation is called a ‘recombinant DNA
molecule’ or a ‘clone’.
• Each clone can be replicated to provide ample material
for study.

CLONING VEHICLES (VECTORS)
Cloning vector is a DNA molecule that carries
foreign DNA into a host cell, replicates inside a
bacterial (or yeast) cell and produces a large
number of copies of itself and the foreign DNA.

FEATURES OF CLONING VECTORS
 A cloning vector should possess an origin of replication so that it
can self-replicate inside the host cell.
 It should have a restriction site for the insertion of the target DNA.
 It should have a selectable marker with an antibiotic resistance
gene that facilitates screening of the recombinant organism.
 It should be small in size so that it can easily integrate into the
host cell.
 It should be capable of inserting a large segment of DNA.
 It should possess multiple cloning sites.
 It should be capable of working under the prokaryotic and
eukaryotic systems.

CLONING VECTORS
Origin of replication: a sequence from where
replication starts.
Selectable marker: a method of selecting for
bacteria containing a vector with foreign
DNA; permits the growth of transformants
and eliminate the non-transformants;
usually accomplished by genes encoding
resistant to antibiotics such as ampicillin,
chloramphenicol, tetracycline or
kanamycin.
Cloning site: to insert foreign DNA; the most
versatile vectors contain a site that can be
cut by many restriction enzymes.

A TYPICAL PLASMID VECTOR
It contains a polylinker
which can recognize
several different
restriction enzymes, an
ampicillin-resistance
gene (ampr) for selective
amplification, and a
replication origin (ORI)
for proliferation in the
host cell.

TYPES OF CLONING VECTORS (PLASMIDS)
 These were the first vectors used in gene
cloning.
 These are found in bacteria, eukaryotes
and archaea.
 These are natural, extrachromosomal, self-
replicating DNA molecules.
 They have a high copy number and possess
antibiotic-resistant genes.
 They encode proteins which are necessary
for their own replication.
 pBR322, pUC18, F-plasmid, R-plasmid are
some of the examples of plasmid vectors.

PLASMID VECTORS
• Plasmid is an independent, circular,
self-replicating DNA molecule that
carries only a few genes.
•They are self replicating because they
have an origin of replication (ori) and
controls the copy number also.
•Plasmids are double-stranded, extra-
chromosomal pieces of DNA.
•They carry antibiotic resistance genes
and a region to clone the ‘new’ DNA
called the multiple cloning site.

PLASMID VECTORS Some other properties
of plasmids:
•They are always double
stranded.
•Circular in shape.
•Associated histone
proteins are absent.
•Introns are absent.
•Replicates independent
of main genome.
•Does not act as genetic
factor.

 These are more efficient than
plasmids for cloning large DNA
inserts.
 Phage λ and M13 phage are
commonly used bacteriophages in
gene cloning.
 53 kb DNA can be packaged in the
bacteriophage.
 The screening of phage plaques is
much easier than the screening of
recombinant bacterial colonies.
BACTERIOPHAGE

 These are artificial vectors.
 They are used in combination with M13 phage.
 They possess multiple cloning sites and an
inducible lac gene promoter.
 They are identified by blue-white screening.
Other cloning vectors include:
 Yeast Artificial Chromosomes (YAC) and
Bacterial Artificial Chromosomes (BAC) .
 Cosmids.
 Retroviral Vectors.
 Human Artificial Chromosomes (HAC).
 Shuttle vectors.
 Transposons as vectors.
PHAGEMIDS (PHASMIDS)

 BACs are similar to E.coli plasmids
vectors.
 It is obtained from naturally
occurring F’ plasmid.
 These are used to study genetic
disorders such as Alzheimer’s
disease or in the case of down
syndrome.
 They can accommodate large DNA
sequences without any risk.
BACTERIAL ARTIFICIAL CHROMOSOMES (BAC)

 YAC is a human engineered DNA
molecule used to clone DNA
sequences in yeast cells.
 YACs are shuttle vectors capable of
replicating and being selected in
common bacterial hosts such as
E.coli as well as in the yeast S.
cerevisiae.
 They are capable of carrying
approximately up to 1000 kbp of
inserted DNA sequence.
YEAST ARTIFICIAL CHROMOSOMES (YAC)

• Agrobacterium tumefaciens can
cause crown gall tumors in dicot
plant by transferring specific
genes.
• A. tumefaciens contains a large
plasmid called Ti plasmid which
can deliver T- DNA to transform
normal cell into a tumor and direct
these tumor cells to produce the
desired chemicals.
• Plant genetic engineers have used
this natural transformation system
as a vehicle for the introduction of
foreign DNA into plants.
VECTORS FOR CLONING GENES IN PLANTS

THE GENOME OF AGROBACTERIUM TUMEFACIENS C58 HAS BEEN SEQUENCED
COMPLETELY AND CONSISTS OF A CIRCULAR CHROMOSOME, A LINEAR
CHROMOSOME AND TWO PLASMIDS
Ti Plasmid
T-DNA region
Virulence region
Opine
catabolism
Tumor-producing
genes
During infection, the Ti plasmid is integrated
into the plant chromosomal DNA

STEPS OF THE AGROBACTERIUM TUMEFACIENS-MEDIATED PLANT
TRANSFORMATION PROCESS
• Attachment of A. tumefaciens to
the plant cells.
• Sensing plant signals by A.
tumefaciens and regulation of
virulence genes in bacteria following
transduction of the sensed signals.
• Generation and transport of T-DNA
and virulence proteins from the
bacterial cells into plant cells.
• Nuclear import of T-DNA and
effecter proteins in the plant cells.
• T-DNA integration and expression
in the plant genome.

 Retroviruses in animals have the ability to transform the
normal cells into cancerous cells.
 Retroviruses are disarmed and now used to deliver
desirable genes into animal cells.
VECTORS FOR CLONING GENES IN ANIMALS

 Cell membrane being hydrophilic in
nature, does not permit DNA to pass
through it.
 Bacterial cell is treated with divalent
cation like calcium chloride CaCl2 to make
them competent.
 Incubate the cells with r-DNA on ice.
 Followed by heat shock by placing them
at 42oC and again putting them back on
ice.
 This enables the bacteria to take up the r-
DNA.
COMPETENT HOST (CHEMICAL TREATMENT)

MICROINJECTION
•Microinjection is a direct physical method
involving the mechanical insertion of the
desirable DNA into a target cell.
•The target cell may be the one identified from
intact cells, protoplasts, callus, embryos,
meristems etc. (it is preferred for animal cells)
•The technique of microinjection involves the
transfer of the gene through a micropipette (0.5-
10.0 pm tip) into the cytoplasm/nucleus of a plant
cell or protoplast.
•While the gene transfer is done, the recipient
cells are kept immobilized in agarose embedding.

GENE GUN TECHNIQUE
Microinjection
vacuum chamber
•The gene gun techniques is known as
the biolistic particle delivery system.
Gold particles (1mm in diameter) are
coated with DNA containing the gene of
interest, and are propelled using the
gene gun into the host plant tissue.
•The particles are able to penetrate the
plant cell walls and deliver the DNA
inside the cell.
GENE GUN

ELECTROPORATION
Electroporation is often used
to transform bacteria, yeast,
or plant protoplasts by
introducing new coding DNA.
Electroporation works by
passing thousands of volts
across a distance of one to
two millimeters of suspended
cells in an electroporation
cuvette (1.0 – 1.25 kV, 250 –
750 V/cm).

 Isolation of the Genetic
Material(DNA)
 Cutting of DNA at specific
location
 Amplication of Gene of Interest
using PCR
 Insertion of r-DNA into the Host
cell/organism
 Obtaining the Foreign Gene
Product
PROCESSES OF RECOMBINANT DNA TECHNOLOGY

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

 Bacterial cell or plant cells or animal cells are
treated by enzymes like lysozyme for bacteria,
cellulase for plant cells and chitinase for
fungus.
 Proteins are removed by using protease.
 RNA are removed by treating with
ribonuclease.
 DNA is precipitated out by using chilled
ethanol.
 Agarose gel electrophoresis is used to check
the progression of restriction enzyme
digestion and to isolate DNA at anode.
ISOLATION OF THE GENETIC MATERIAL(DNA)

 To separate DNA using agarose
gel electrophoresis, the DNA is
loaded into pre-cast wells in the
gel and a current applied.
 The phosphate backbone of the
DNA (and RNA) molecule is
negatively charged, therefore
when placed in an electric field,
DNA fragments will migrate to
the positively charged anode.
ISOLATION OF DNA (AGAROSE GEL
ELECTROPHORESIS)

Break down the
cell wall and
membranes
Centrifuge to
separate the
solids from the
dissolved DNA
Precipitate
the DNA
using
isopropanol
Centrifuge to
separate the
DNA from the
dissolved salts
and sugars
Wash the
DNA pellet
with Ethanol
and dry the
pellet
Isolated DNA
DNA Extraction

Steps Involved in PCR
 1. Denaturation by
heat.
 2. Annealing Primer to
target sequence.
 3. Extension.
 4. End of the first PGR
cycle.
AMPLICATION OF GENE OF INTEREST USING PCR

STEPS INVOLVED IN POLYMERASE CHAIN REACTION (PCR)
 PCR is divided into three steps:
 Denaturation- The dsDNA becomes single-stranded or denature at a higher
temperature (960 C), during denaturation the hydrogen bonds between two
DNA strands break.
 Annealing- The primer binds or anneals to its exact complementary
sequence on a single-stranded template DNA when cooled up to 55-65°C. The
primer provides a site for the initiation of synthesis.
 Extension- Raise the reaction temperatures to 72°C so Taq polymerase
extends the primers and starts DNA synthesis by adding nucleotides to the
growing DNA strand. Taq DNA polymerase uses the 3’ end of the primer.
 All three steps are repeated for 25 to 40 cycles and in each cycle the DNA
becomes double.

INSERTION OF R-DNA INTO THE HOST CELL/ORGANISM
There are multiple ways foreign DNA can be
introduced into cells including transformation,
transduction, conjugation, and transfection.
•Transformation is the uptake of genetic material
from the environment by bacterial cells. This is
commonly done using calcium chloride which
permeabilizes the cell membrane so the bacteria
can easily uptake the plasmid of interest.
•Transduction occurs when foreign DNA or RNA
is introduced into bacterial or eukaryotic cells via
a virus or viral vector. To do this scientists
commonly use Phagemids, a DNA cloning vector
that contains both bacteriophage and plasmid
properties.

 During conjugation, genetic material
is transferred from a donor
bacterium to a recipient bacterium
through direct contact.
 Transfection is the process by which
foreign DNA is deliberately
introduced into a eukaryotic
cell through non-viral
methods including both chemical
and physical methods e.g.
electroporation or microinjection.
INSERTION OF R-DNA INTO THE HOST CELL/ORGANISM

INSERTIONAL INACTIVATION TECHNIQUE
Insertional inactivation technique of recombinant DNA technology
used to select bacteria that carry recombinant plasmids.
A fragment of foreign DNA is inserted within the coding sequence
of an enzyme β-galactosidase in the presence of a chromogenic
substrate which results into inactivation of the enzyme.

 For example, insertion of a piece of
foreign DNA into a cloning site which
is located on an antibiotic-resistant
gene on the vector can lead to loss of
the antibiotic resistance phenotype
by insertional inactivation.
 The recombinant vector will,
therefore, specify antibiotic
sensitivity, while the non-
recombinant vector will specify
antibiotic resistance.
INSERTIONAL INACTIVATION TECHNIQUE

 The desired (recombinant) proteins such as antibiotics and
insulin etc. need to be produced in large scale.
 To produce this product in large quantity bioreactors are
needed.
 In such reactors 100-1000 litres of culture can be processed.
 They are sterile large vessels with gassing facilities required
for starting a biochemical reaction.
 Control over temperature, moisture, pH level, oxygen levels
and stirring rate will yield the most suitable conditions
necessary for maximized cell growth and productivity.
 Commonly used bioreactors are Stirred Tank type, Bubble
Column type, Fluidized Bed type, Airlift type, Packed Bed
type, sparged stirred tank and Photo Bioreactors, etc.
OBTAINING THE FOREIGN GENE PRODUCT

The basic design of stirred – tank bioreactor
constitutes the following components:
 An agitator system
 An oxygen delivery system
 A foam control system
 A temperature control system
 A pH control system
 Sampling ports
 A cleaning and sterilization system
 A sump and dump line for emptying of the reactor

 In sparged stirred-tank bioreactor sterile air is sparged through
the reactor. The bioreactor has an agitator system, an oxygen
delivery system and a foam control system, a temperature
control system, pH control system and sampling ports so that
small volumes of the culture can be withdrawn periodically.

 The Bubble Column Reactors represent
contactors in which a gas or a mixture of gases
is distributed in the liquid at
the column bottom by an appropriate
distributor and moves upwards in the form
of bubbles causing intense mixing of the liquid
phase.
 The flow rate of air/gas influences the
performance factors- O2 transfer, mixing.
 The Bubble Column Bioreactors may be fitted
with perforated plates to improve
performance.
Bubble Column Reactor

 Fluidized-bed reactors (FBR) are the
most popular reactor configurations
employed for reactions involving solid
reactants.
 In the FBR, a fluidization medium (gas
or liquid) is passed through the bed of
solid reactants at high enough
velocities to suspend the solid and
cause it to behave like a fluid.

•Airlift bioreactors are tower
reactors for large-scale aerobic
cultures where the mixing of the
culture broth is done by the
inserted gas via an airlift pump .
• This pump injects compressed air
at the bottom of the discharge
pipe, which is immersed in the
liquid.

THANK YOU
.
V.S.Malik
Biology Lecturer (Retd.)

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