CHAPTER-09BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Biotechnology Definition:
Biotechnology involves using living organisms or their enzymes to create products beneficial to
humans. Examples: Curd, bread, wine (traditional), and modern techniques such as genetically
modified organisms, hormones, vaccines, gene therapy.
The European Federation of Biotechnology (EFB) defines biotechnology as “The integration of
natural science and organisms, cells, parts thereof and molecular analogues for products and
services”.
Principles of Biotechnology:
1. Genetic engineering − Technique to introduce foreign genetic material (DNA/RNA) into the host’s
genome and altering its phenotype.
2. Maintenance of sterile (microbial contamination free) ambience in chemical engineering processes
for manufacture of products such as protein, antibiotics, vaccines, etc

Basic steps in recombinant DNA

technology.

a) Identification of DNA with desirable genes:

Gene of interest (e.g., disease resistant gene).

b) Introduction of the identified DNA into the

host: A vector DNA such as plasmid is used to
deliver an alien (foreign gene /Gene of
interest) piece of DNA into the host organism.

c) Maintenance of introduced DNA in the host

and transfer of the DNA to its progeny:

Construction of First Recombinant DNA Molecule

 First recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert Boyer (1972).
 They isolated an antibiotic resistance gene (piece of DNA) from a plasmid of Salmonella
typhimurium.
 It was linked with a plasmid vector and transferred into E. coli
 As a result, the gene was expressed & multiplied in E. coli and makes the [Link] cell
resistant against that antibiotic.
Tools of Recombinant DNA Technology
Restriction Enzymes:
Discovered in 1963; enzymes that cut DNA at specific sequences.
Restriction enzymes belongs to Nucleases and are of two kinds:
Endonucleases − Cut the DNA at specific positions within the Restriction sites.
Exonucleases − Cut the DNA at the ends (Remove the nucleotides at the ends of the DNA)

Over 900 restriction enzymes have been isolated, all of which recognize different DNA sequences.
(Recognition sequences) These sequences are Pallindromic in DNA.

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Restriction enzymes cut a little away from the center of palindrome site, but between the same two
bases on the opposite strands. As a result, overhangs (called sticky ends) are generated on each

strand. ● Sticky ends form hydrogen bonds with their complementary counter parts with help of DNA
ligases to form rDNA molecule.
E.g., Palindromic nucleotide sequence for EcoRI (cleaves the H-H bonds between the bases G-A of
the palindrome sites GAATTC of both the strands) is
5‘ —— GAATTC —— 3‘
3‘ —— CTTAAG —— 5‘

RECOMBINANT DNA MOLECULE

Naming/Nomenclature of the Restriction enzymes:

First letter indicates the first letter of Genus.
The second two letters indicate species. The third letter indicates the first letter of strain
Roman numbers is the order in which the enzymes were isolated from that strain of bacteria).
“EcoRI’ is isolated from the bacteria Escherichia coli
E from Escherichia, co from coli, R from ‘RY13’, ‘I’ isolated first from that bacteria

1. Origin of Replication (ori): Initiates replication, controls copy

number.
2. Selectable Markers: Identify transformed cells, e.g.,
antibiotic resistance genes.
3. Cloning Sites: Restriction sites for inserting
foreign DNA.

Cloning Vectors:
 Plasmids and Bacteriophages: Capable of replicating independently within bacterial cells.
Vectors for Plants and Animals:
[Link] tumefaciens (Ti plasmid) for plants, 2. Retroviruses for animals.

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Competent Host: (For Transformation with Recombinant DNA)
Method Explanation
Divalent Cation Treating a cell with a specific concentration of a divalent Cation,
such as calcium, increases the cell’s efficiency to take DNA.
Heat and shock incubating the host cell under heat and then ice, create pores on
the wall through which DNA enters the bacterium.
Micro-injection rDNA is directly injected into the nucleus of an animal cell
Biolistic/ Gene gun method plant cells are bombarded with high-velocity micro-particles of
gold or tungsten coated with DNA.
Disarmed pathogen vectors Infect host cell and transfer r- DNA into them
Processes of Recombinant DNA Technology
• Isolation of Genetic Material Breaking cells open to release DNA using enzymes like
(DNA): lysozyme (bacteria), cellulase (plants), chitinase (fungi).
Removing RNA and proteins to purify DNA.
• Cutting of DNA at Specific Using restriction enzymes to cut DNA at specific sites.
Locations: Visualizing DNA fragments using agarose gel
electrophoresis.
• Amplification of Gene of Interest In vitro synthesis of multiple DNA copies using PCR
using PCR:
• Insertion of Recombinant DNA into Introducing ligated DNA into host cells to transform them,
Host Cells: e.g., E. coli with antibiotic resistance genes.
Selecting transformed cells using selective media.
• Obtaining the Foreign Gene Cloning genes into vectors and transferring into host cells
Product: for expression.
Producing recombinant proteins in large scale using
bioreactors
• Downstream Processing: Purification and formulation of the product after
biosynthesis.
Clinical trials and quality control testing.
Gel Electrophoresis
a) The fragments of DNA obtained after cutting
with restriction enzymes are separated by
using gel electrophoresis.
b) Electric field is applied to the electrophoresis
matrix (commonly agarose gel) and
negatively charged DNA fragments move
towards the anode.
c) Fragments separate according to their size e) DNA fragments are visible as bright orange
by the sieving properties of agarose gel. coloured bands in the agarose matrix.
Smaller the fragment, farther it moves. f) These bands are cut from the agarose gel
d) Staining dyes such as ethidium bromide and extracted from the gel piece (elution).
followed by exposure to UV radiations are g) DNA fragments are purified and these
used to visualize the DNA fragments. purified DNA fragments are used in
constructing recombinant DNAs.

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 Polymerase Chain Reaction (PCR)
PCR consists of 3 steps:
Denaturation Double helical DNA is denatured by providing high temperature (94 ᵒC). DNA
polymerase does not get degraded in such high temperatures since the DNA
polymerase used in this reaction is thermostable as it is isolated from
thermophilic bacteria, Thermus aquaticus (Taq).
Annealing Joining of the two Primers at the 3’ end of DNA template (at about 52 ᵒC).
Extension Addition of nucleotides to the primer and extending the chain (at about 72 ᵒC).
This cycle is repeated several times to generate up to 1 billion identical copies of the DNA.

DENATURATION

ANNEALING

EXTENSION

Bioreactors
 These are the vessels in which raw materials are biologically converted to specific products,
enzymes etc., using microbial, plant, animal, or human cells.
 Bioreactors are used to produce large quantities of products. They can process 100-1000
litres of culture.
 A bioreactor provides the optimal growth conditions (pH, temperature, substrate, salts, and
vitamins, oxygen) to get desired product.
 The most used bioreactors are of stirring type (stirred-tank bioreactor). 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 the following facilities.
 1. An agitator system 2. An oxygen delivery system
 3. A foam control system 4. A temperature control system
 5. pH control system 6. Sampling ports (for periodic withdrawal of the culture)

Downstream Processing: – It is a series of processes such as separation and purification of

products after the biosynthetic stage.
– The product is formulated with suitable preservatives. Such formulation undergoes thorough
clinical trials and strict quality control testing.

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