Biotechnology : Principles & Processes

Introduction
Biotechnology deals with the techniques of using live organisms or enzymes from organisms to produce
products & processes useful to humans.

Parameter Traditional biotechnology Modern biotechnology

Organisms involved Microbes Genetically modified organisms

Production Small scale Large scale

Examples/Technique
Curd, bread or wine making In vital fertilisation leading to
include
a ‘test-tube’ baby

Note Synthesising a gene & using it

EFB (European Federation of Biotechnology) Developing a DNA vaccine
'The integration of natural science & organisms, cells, parts thereof, & molecular Correcting a defective gene
analogues for products & services'.
It encompasses both traditional view & modern molecular biotechnology.

Principles of Biotechnology/Core technique involved in Modern Biotechnology

Parameters Genetic engineering - Bioprocessess engineering

Techniques to alter chemistry of Maintenance of sterile ambience
Definition in chemical engineering processes
genetic material to introduce these
into host organisms, & thus change to enable growth of only the
the phenotype of host organisms desired microbes/eukaryotic cell in
large quantities
 Include Creation of rDNA Manufacturer of Biotechnology
Gene cloning products like antibiotic,
Gene transfer vaccines , enzymes, etc.

The ability to multiply copies of antibiotic resistance gene in E .coli was called cloning of antibiotic resistance gene
Notes in E .coli

Advantages of Biotechnology over other techniques

Methods Advantage Disadvantage

Preserves genetic information No variations

I. Asexual reproduction

II. Sexual reproduction opportunities for variations & formulation of Some of which may be harmful to the
unique combinations of genetic setup. organism as well as the population

Very often to inclusion &

III. Traditional hybridisation Used in plant & animal breeding. multiplication of undesirable gene
along with desirable genes.

IV. Genetic engineering Allows us to isolate & introduce only one or a

set of desirable genes without introducing ——
undesirable genes into target organism.

3 basic steps in Genetically Modifying Organisms

1) Identification of DNA with desirable genes

2) Introduction of the identified DNA into the host
3) Maintenance of introduced DNA in the host & transfer of the DNA to its progeny
 Key tools of recombinant DNA technology

1) Enzymes 2) Vectors 3) Competent host cells

Enzymes - Nucleases
Mostly commonly used enzymes in genetic engineering are DNA polymerase
Nucleases - Ligases
Catalyse the cleavage of nucleic acids.
Type

Exonucleases Endonucleases

Remove nucleotides from the Make cuts at specific positions within the DNA i.e.
ends of the DNA at recognition/palindromic sequence

In the year 1963, the two enzymes responsible for restricting the growth
of bacteriophage in Escherichia coli were isolated

Methylase Restriction endonuclease/ Molecular scissors

Add methyl groups to bacterial DNA Cut the DNA of bacteriophage

Enzymes
Restrictions endonuclease
More than 900 restrictions enzymes have been isolated from over 230 strains of bacteria (prokaryotic cell) each
of which recognise different recognition sequences.
Nomenclature/Naming of enzymes:

Functions by:
‘Inspecting’ the length of DNA sequence
Binds to the “specific recognition sequence “
Cuts the two strands of dsDNA at specific points in their
sugar-phosphate backbones and leaves single stranded
portions at the ends.
These overhanging stretches & called sticky ends.

Ligase
When source DNA & vector DNA are cut by the same restriction enzyme the resultant DNA fragments have
the same kind of ‘sticky-ends’. Sticky ends are named so because they form hydrogen bonds with their
complementary cut counterparts & this stickiness facilitates the action of the enzyme DNA Ligase.

Note:
First restriction endonuclease - HindII : Isolated & characterised five years later, recognises sequence of 6bp.
First recombinant DNA was prepared by Stanley Cohen & Herbert Boyer, 1972:

Antibiotic resistance gene Introduced

Recombinant plasmid E.coli
Plasmid of Salmonella typhimurium Into
 Cloning Vectors

Vectors are vehicles for delivering foreign DNA into recipient cells.
Vectors used at present are engineered in such a way that they help easy linking of foreign DNA & selection of
recombinant from non recombinants
Features of cloning vectors:

1) Origin of Replication (ori):

Sequence from where replication starts
Responsible for controlling copy number of the linked DNA
Those vectors are preferred which support high copy number
2) Selectable Marker:
Helps in selection of transformants
Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol,
tetracycline or kanamycin, etc., are considered useful selectable markers for E.coli.
The normal E.coli cells do not carry resistance against any of these antibiotics
3) Cloning sites/Restriction Sites
Single recognition site for a restriction enzyme within the vector is a preferable feature.
Presence of more than one recognition sites within the vector will generate several
fragments, which will complicate the gene cloning
The ligation of alien DNA/gene of interest (GOI) is carried out a restriction site present
in one of the antibiotic resistant genes.
Transformation: Procedure through which piece of foreign DNA is introduced in a host bacterium.
Insertional inactivation: Insertion of GOI within antibiotic resistance gene/selectable marker results in
inactivation/formation of the coded product.
Hypothesis: Inseration of GOI at Bam HI site in tet.
If transformation fails - Non transformants are obtained in antibiotic lacking agar medium but they
don’t grow on antibiotic rich medium.
If transformation sucessful - Transformants obtained are of two types:

All transformants are not

Non Recombinants Recombinants
Recombinants but all
recombinants are Insertional inactivation

transformants. Ligate
One antibiotic resistant gene
helps in selecting the
transformants whereas the Foreign DNA
other antibiotic resistant gene at Bam HI site
helps in selection of
recombinant Gene of interest cloned
Resistance to ampicillin

Resistance to tetracycline
(due to inactivation)

rop —> codes for the proteins involved in the replication of the plasmid
 Plasmid as vectors:
Extra chromosomal, circular, double stranded DNA.
Replicate independent of the control of chromosomal DNA (autonomously).
They may have 1 or 2 copies per cell or even 15 - 100 copies per cell.

OTHER CLONING VECTORS

Selection of Recombinants due to inactivation of antibiotic resistance gene as in pBR322 is a
cumbersome procedure because it requires simultaneous plating of two plates having different
antibiotics.

To overcome the disadvantages of pBR322, alternative selectable markers (lazy Z) acting as reporter
enzyme have been developed which differentiate Recombinants from non-Recombinants on the basis of
their ability to produce colour in the presence of chromogenic substrate.

lac Z gene coding for beta-galactosidase acts as selectable marker in the plasmid
Experiment: Insert foreign DNA at lac Z gene + transformations in E.coli
Chromogenic substrate

Fails Succeeds

Blue coloured colonies White coloured colonies

Non-Recombinants Recombinant

Ti plasmid of Agrobacterium tumefaciens

Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA know as ‘T-DNA’ to
transform normal plant cells into a tumor & direct the tumor cells to produce the chemicals required by the pathogen.
Disarmed tumour inducing (Ti) plasmid is used which is no more pathogenic to the plants but is still able to use the mechanism to deliver
the genes of our interest into varieties of plants.

Bacteriophages
High copy number than plasmid

Retroviruses

Retroviruses in animals have the ability to transform normal cells into cancerous cells
Disarmed retroviruses are used to deliver desirable genes into animals cells

METHODS OF TRANSFORMATION
I. Micro - injection
Recombinant DNA is directly injected into the nucleus of an animal cell.

II. Biolistic/Gene gun

Plant cells are bombarded with high velocity micro particles of gold or
tungsten coated with DNA.

III. Heat shock method

IV. “Disarmed pathogens “ vector

 COMPETENT HOST FOR TRANSFORMTION WITH RECOMBINANT DNA

DNA is hydrophilic, so it can not pass through cell membranes

In order to force cell to take up alien DNA/rDNA, it must first be made ‘competent’ by treating with ice cold
calcium chloride.
Entry of rDNA in host cell is due to transient pores created by heat shock (42 degree Celsius)
and not due to calcium ions.
Divalent cations increase the efficiency with which DNA enters the bacterium through pores in its cell wall.

PROCESS OF RECOMBINANT DNA TECHNOLOGY

Isolation of DNA

Fragmentation of DNA by restriction endonuclease

Isolation of desired DNA fragments (electrophoresis)

Amplification of gene of interest (PCR)

Ligation of the DNA fragment into a vector

Transferring the alien DNA/recombinant DNA into the host

Culturing the host cells in a medium at large scale (Bioreactors)

Extraction & purification of the desired product

I. Isolation of Genetic Material (DNA)

In majority of organisms, DNA is the genetic material

Since DNA is enclosed within the membranes, we have to break the cell open to release DNA along with
other macromolecules
 Macromolecules Bacteria Lysozyme
Fungi Chitinase

Plant cell Cellulase

In order to get DNA in pure form (free form other macromolecules), it is treated with different enzymes like RNase,
protease etc.

Process
Centrifuge Chilled ethanol to precipitate DNA Spooling

Pure DNA

II. Fragmentation by restriction endonucleases

III. Separation & isolation of DNA fragments

Gel electrophoresis

Separation of negatively charged DNA molecules under an electric field through a

medium/matrix.
Most commonly used matrix for DNA separation is
Natural polymer, obtained from sea weeds
Agarose
Separate DNA fragments through seiving effect

— cathode
+ anode

Stained with Exposed to

Gel Ethidium Bromide U.V rays Bright organs bands

Elution Process Removal of DNA fragments from gel

Note
Purified DNA fragments are generally amplified (PCR) before constructing rDNA joining
with cloning vector.

IV. PCR - Polymerase Chain Reaction

In vitro amplification of DNA (gene of interest)

Reaction mixture Work/Function

Nucleotide Formation of DNA chain

2 sets of chemically synthesised

Primers oligonucleotides, complementary to the
regions of DNA.

Thermostable DNA polymerase, isolated from

bacterium, Thermus aquaticus, remains
Taq polymerase
active during high temperature induced
denaturation of dsDNA. It extends the
primers i.e. meant for chain elongation.

Genome DNA Template DNA for gene of interest

Sequence of events

The amplified fragments if desired can now be used to ligate with a vector for further
cloning.
 V. Ligation of the DNA fragments into a vector by DNA Ligase

VI. Insertion of recombinant DNA into the host cell

Transformed host cell are selected with the help of selectable marker genes.
VII. Culturing of recombinant host cells (Biosynthetic stage)

The cells harbouring cloned genes of interest may be grown in laboratory/

bioreactors
Note

Bioreactors: Vessels in which raw materials are biologically converted into specific products
using microbial plant, animal human cells & provide optimal growth conditions (temperature, pH,
substrate, salts, vitamins, oxygen)
 Parameters Laboratory Bioreactors

Culture Large volumes (100 - 1000 lts)

Small volume

Maintaining optimal conditions Not possible

Growth rate of cell Never optimal

Optimum

Production Small scale Large scale

Cylinderical or with curved base Facilitate mixing of reactor content

Commonly used Bioreactors Facilitate even mixing & oxygen

Stirrer
are stirred type having availability throughout the bioreactor
Agitator system

Oxygen delivery system

pH control system
Foam control system

Sampling ports To withdraw small volumes of culture

periodically
Types of stirred tanks

Simple stirred tank Sparrged stirred tank Note

In open culture system/ continuous culture sytem

Used medium is drained out from one side while fresh medium is
added from the other to maintain the cells in their
physiologically most active log/exponential phase.

Larger biomass —> Higher yields of desired protein.

VIII. Downstream processing
Separation & purification of the desired product/recombinant protein from heterologous
host (non native host).

Product has to be formulated with suitable preservatives

Strict quality control testing is done for each product

The downstream processing & quality control testing vary from product to
product

IX. Product is subjected for marketing as a finished product