Biotechnology: Principles and Processes 7

Chapter

1 INTRODUCTION 2 PRINCIPLES OF BIOTECHNOLOGY/CORE TECHNIQUES INVOLVED

IN MODERN BIOTECHNOLOGY
m Biotechnology deals with techniques of using live organisms or enzymes from
organisms to produce products and processes useful to humans.
Parameters Genetic engineering Bioprocess engineering
m Definition m Techniques to alter the chemistry of m Maintenance of sterile ambience in
genetic material to introduce these chemical engineering processes to
Parameters Traditional biotechnology Modern biotechnology into host organisms, and thus change enable growth of only the desired
m Organisms involved m Microbes m Genetically modified the phenotype of host organism microbe/eukaryotic cell in large
organisms quantities
m Production m Small scale m Large scale m Include m Creation of rDNA m Manufacture of biotechnological
m Examples/Technique m Curd, bread or wine m In vitro fertilisation m Gene cloning products like antibiotics, vaccines,
include making leading to a ‘test -tube’ m Gene transfer enzymes, etc.
baby
The ability to multiply copies of antibiotic resistanse gene in E.coli was called cloning of antibiotic
EFB (European Federation of Biotechnology) m Synthesising a g e n e resistance gene in E.coli.
m ‘The integration of natural science and and using it
organisms, cells, parts thereof, and molecular m D e v e l o p i n g a D N A
analogues for products and services’. vaccine 4 THREE BASIC STEPS IN GENETICALLY MODIFYING ORGANISMS
m It encompasses both traditional view and m Correcting a defective (1) Identification of DNA with desirable genes (2) Introduction of the identified DNA into the host
modern molecular biotechnology. gene (3) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny

3 ADVANTAGES OF BIOTECHNOLOGY OVER OTHER TECHNIQUES 5 KEY TOOLS OF RECOMBINANT DNA TECHNOLOGY

Methods Advantage Disadvantage (1) Enzymes (2) Vectors (3) Competent host cells Nucleases
I. Asexual reproduction Preserves genetic No variations Enzymes - Most commonly used enzymes in genetic engineering are DNA polymerase
information
Ligases
II. Sexual reproduction Provides opportunities for Some of which may be Nucleases - Catalyse the cleavage of nucleic acids.
Types
variations and formulation harmful to the organism as
of unique combinations of well as the population
genetic setup Exonucleases Endonucleases
Palindromic sequence
III. Traditional Used in plant and animal Very often lead to inclusion Remove nucleotides Make cuts at specific positions within the DNA reads same on the two
hybridisation breeding. and multiplication of from the ends of the DNA i.e. at recognition/palindromic sequence
strands (from 5¢ ® 3¢ and
undesirable genes along 3¢ ® 5¢ direction) when
with desirable genes. m In the year 1963, the two enzymes responsible for restricting the orientation of reading is
growth of bacteriophage in Escherichia coli were isolated kept same
IV. Genetic engineering Allows us to isolate and
—
introduce only one or a set
of desirable genes without
introducing undesirable Methylase Restriction endonuclease / Molecular scissors
genes into target organism. Add methyl groups to bacterial DNA Cut the DNA of bacteriophage
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6 ENZYMES
m Restriction endonuclease I II III IV m Ligase
Eco RI
More than 900 restriction enzymes have been isolated l When source DNA and vector DNA are cut by the same restriction enzyme the
Genus Species Strain Order of
from over 230 strains of bacteria (prokaryotic cell) each Escherichia coli RY13 isolation resultant DNA fragments have the same kind of ‘sticky-ends’. Sticky ends are named
of which recognise different recognition sequences.
so because they form hydrogen bonds with their complementary cut counterparts and
l Nomenclature/Naming of enzyme :
this stickiness facilitates the action of the enzyme DNA ligase.
l Functions by: Vector DNA Foreign DNA
Sticky end

· ‘Inspecting’ the length of DNA sequence

· Binds to the “specific recognition sequence”
Sticky end
m First restriction endonuclease - Hind II : Isolated and characterised five years
later, recognises sequence of 6 bp.
· Cuts the two strands of ds DNA at specific points in Ligase
their sugar-phosphate backbones a n d l e a v e s m First recombinant DNA was prepared by Stanley Cohen and Herbert Boyer, 1972 :
single stranded portions at the ends. l Antibiotic resistant gene Introduced
Recombinant plasmid Escherichia
· These overhanging stretches and called s t i c k y l Plasmid of Salmonella into
Recombinant DNA coli
typhimurium
ends. Steps in formation of rDNA

7 CLONING VECTORS

m Vectors are vehicles for delivering foreign DNA into recipient cells. m Transformation: Procedure through which piece of foreign DNA is introduced in a host
m Vectors used at present are engineered in such a way that they help easy linking of bacterium.
l Insertional inactivation: Insertion of GOI within antibiotic resistance gene/selectable marker
foreign DNA and selection of recombinants from non recombinants
results in inactivation/formation of the coded product.
Features of cloning vectors: R
l Hypothesis: Insertion of GOI at Bam HI site in tet .
(1) Origin of Replication (ori): l If transformation fails – Non transformants are obtained in antibiotic lacking agar medium but
m Sequence from where replication starts they don’t grow on antibiotic rich medium.
l If transformation successful – Transformants obtained are of two types:
m Responsible for controlling copy number of the linked DNA
m Those vectors are preferred which support high copy number m All transformants are not
recombinants but all Non Recombinants Recombinants
(2) Selectable Marker: Insertional
recombinants are EcoR I Cla I Hind III
inactivation
m Helps in selection of transformants transformants. Pvu I
Pst I BamH I
Normally, the genes encoding resistance to antibiotics such as ampicillin, Ligate
m m One antibiotic resistant gene helps amp
R
tet
R R R
Foreign amp tet
chloramphenicol, tetracycline or kanamycin, etc., are considered useful in selecting the transformants E.coli cloning pBR322 Sal I DNA at
selectable markers for E.coli whereas the other antibiotic vector pBR322
ori
Bam HI
rop site
m The normal E.coli cells do not carry resistance against any of these resistant gene helps in selection of
antibiotics recombinants
Pvu II
m rop ® codes for the proteins Gene of interest cloned û ü
(3) Cloning Sites/Restriction Sites
involved in the replication of the Resistance to ampicillin ü ü
m Single recognition site for a restriction enzyme within the vector is a plasmid Resistance to tetracycline ü û
preferable feature. [due to inactivation]
m Presence of more than one recognition sites within the vector will generate m Plasmids as vectors:
several fragments, which will complicate the gene cloning l Extra chromosomal, circular, double stranded DNA.

m The ligation of alien DNA/gene of interest (GOI) is carried out at a restriction site l Replicate independent of the control of chromosomal DNA (autonomously).
present in one of the antibiotic resistant genes. l They may have 1 or 2 copies per cell or even15 - 100 copies per cell.
 Biotechnology: Principles and Processes 125

9 OTHER CLONING VECTORS 10 METHODS OF TRANSFORMATION I. Isolation of the Genetic Material (DNA)
m In majority of organisms, DNA is the genetic material
Selection of recombinants due to inactivation of antibiotic I. Micro - injection
m Since DNA is enclosed within the membranes, we
resistant gene as in pBR322 is a cumbersome procedure l Recombinant DNA is directly injected into the nucleus of
because it requires simultaneous plating of two plates have to break the cell open to release DNA along with
an animal cell.
having different antibiotics. other macromolecules Bacteria ® Lysozyme
II. Biolistic/Gene gun Fungi ® Chitinase
To overcome the disadvantage of pBR322, alternative Plant cell ® Cellulase
l Plant cells are bombarded with high velocity micro- m In order to get DNA in
selectable markers (lac Z) acting as reporter enzyme have particles of gold or tungsten coated with DNA. pure form (free from other macromolecules), it is
been developed which differentiate recombinants from non-
III. Heat shock method treated with different enzymes like RNase, protease
recombinants on the basis of their ability to produce colour in
IV. “Disarmed pathogen” vector etc.
the presence of chromogenic substrate.
m lac Z gene coding for b-galactosidase acts as selectable Chilled ethanol
11 COMPETENT HOST FOR TRANSFORMATION WITH Pure ® Centrifuge to precipitate DNA
marker in the plasmid DNA

m Experiment: Insert foreign DNA at lac Z gene +
transformation in E.coli
Chromogenic substrate
Fails Succeeds
Blue coloured colonies White coloured colonies
Non-recombinants Recombinants
Process
RECOMBINANT DNA
m DNA is hydrophilic, so it can not pass through cell membranes
Spooling
m In order to force cell to take up alien DNA/rDNA, it must first be
made ‘competent’ by treating with ice cold calcium chloride.
m Entry of rDNA in host cell is due to transient pores created by II. Fragmentation by restriction endonucleases
+2 III. Separation and isolation of DNA fragments
heat shock (42°C) and not due to Ca ions.
m Gel electrophoresis
m Divalent cations increases the efficiency with which DNA
l Separation of negatively charged DNA molecules
enters the bacterium through pores in its cell wall.
under an electric field through a medium/matrix.
l Most commonly used matrix for DNA separation is
12 PROCESS OF RECOMBINANT DNA TECHNOLOGY

m Ti plasmid of Agrobacterium tumefaciens Isolation of DNA Natural polymer, obtained from sea weeds
Agarose
l Agrobacterium tumefaciens, a pathogen of several ¯ Separate DNA fragments through seiving effect
dicot plants is able to deliver a piece of DNA known Fragmentation of DNA by restriction endonucleases Separation on the basis of size
Wells filled with (Smaller the DNA fragment farther it moves)
as ‘T-DNA’ to transform normal plant cells into a DNA fragments DNA
¯ Largest bands
tumor and direct the tumor cells to produce the Smallest
Isolation of desired DNA fragment (electrophoresis) – electrode/ + electrode/

4
chemicals required by the pathogen. cathode anode

3
¯

2
l Disarmed tumour inducing (Ti) plasmid is used

1
which is no more pathogenic to the plants but is still Amplification of gene of interest (PCR)
able to use the mechanism to deliver the genes of ¯ Stained Exposed
with to
our interest into varieties of plants. Gel Ethidium U.V rays

Appears
Ligation of the DNA fragment into a vector
m Bacteriophages Bromide
¯
l High copy number than plasmid Process
Transferring the alien DNA/recombinant DNA into the host Elution Removal of DNA Bright orange
m Retroviruses bands
¯ fragment from gel
l Retroviruses in animals have the ability to transform
normal cells into cancerous cells Culturing the host cells in a medium at large scale (Bioreactors)
l Disarmed retroviruses are used to deliver ¯ m Purified DNA fragments are generally amplified (PCR)
desirable genes into animal cells Extraction and purification of the desired product before constructing rDNA by joining with cloning vector.
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IV. PCR - Polymerase Chain Reaction VII. Culturing of recombinant host cells (Biosynthetic stage) l Bioreactors: Vessels in
m In vitro amplification of DNA (gene of interest) m The cells harbouring cloned genes of interest may be grown in which raw materials are
Laboratory/ Bioreactors biologically converted into
Reaction mixture Work/Function specific products using
Parameters Laboratory Bioreactors microbial plant, animal
Nucleotides Formation of DNA chain human cells and provide
Culture Small volume Large volumes (100 - 1000 lts) optimal growth conditions
Primers 2 sets of chemically synthesised
Maintaining optimal conditions Not possible ü (temperature, pH,
oligonucleotides, complementary to the substrate, salts, vitamins,
regions of DNA Growth rate of cell Never optimal Optimum oxygen)
Taq polymerase Thermostable DNA polymerase, isolated Production Small scale Large scale
from bacterium, Thermus aquaticus, Cylindrical or with curved base Facilitate mixing of reactor contents
remains active during high temperature
m Commonly used Bioreactors Stirrer Facilitate even mixing and oxygen
induced denaturation of dsDNA. It extends availability throughout the bioreactor
are stirred type having Agitator system
the primers i.e. meant for chain elongation.
Oxygen delivery system
Genome DNA Template DNA for gene of interest
pH control system
m Sequence of events Foam control system
Region to be amplified Sampling ports To withdraw small volumes of culture
5¢ 3¢ periodically
ds DNA Steps m Types of stirred tanks
3¢ 5¢ ¯
Heat Denaturation
In Open Culture System/
5¢ 3¢
3¢ 5¢ Simple stirred tank Sparged stirred tank Continuous Culture System
Primers Annealing l Used medium is drained out
5¢ 3¢
3¢ 5¢ Increased from one side while fresh
DNA polymerase
surface medium is added from the other
Motor area for to maintain the cells in their
(Taq polymerase) Acid/Base oxygen Gas
+ deoxynucleotides for pH Foam transfer physiologically most active
5¢ 3¢ entrainment
control braker log/exponential phase.
3¢ 5¢ Extension Flat bladed l Larger biomass ® Higher yields
Stream for
5¢ 3¢ impeller
sterilisation of desired protein.
3¢ 5¢ Culture
broth Bubbles/
30 cycles/Process repeated ‘n’ times dramatically
increase the
oxygen
Amplified Sterile Air
transfer area
(~1 billion times)

VIII. Downstream processing

m The amplified fragment if desired can now be used to ligate
with a vector for further cloning. m Separation and purification of the desired product/recombinant protein from heterologous host (non native
V. Ligation of the DNA fragment into a vector by DNA ligase host).

¯ m Product has to be formulated with suitable preservatives.

VI. Insertion of recombinant DNA into the host cell m Strict quality control testing is done for each product
l Transformed host cells are selected with the help of m The downstream processing and quality control testing vary from product to product.
selectable marker genes. IX. Product is subjected for marketing as a finished product
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1. The definition given by EFB for (3) Introducing both desirable and 8. Choose the odd one w.r.t. ends produced
biotechnology is [NCERT Pg. 193] undesirable genes by Eco RI [NCERT Pg. 197]
(1) Techniques of using live organisms only (4) Being easy to perform (1) Sticky ends
(2) Techniques of using enzymes only to 5. First recombinant DNA was made by linking
(2) Single stranded portion on each strand
produce products and processes useful A with a B of C . Choose the
of DNA
to humans option which correctly fill the blanks A, B
and C. [NCERT Pg. 194] (3) Overhanging stretches on each strand
(3) The integration of natural science and
organisms, cells, parts there of and of DNA
A B C
molecular analogues for product and (4) Blunt ends
(1) Antibiotic Plasmid Salmonella
services resistant typhimurium 9. Restriction endonucleases cut which
(4) Techniques which include only gene bonds? [NCERT Pg. 196]
synthesising a gene and using it (2) Antibiotic Plasmid Escherichia
(1) Hydrogen bonds
2. The core technique of biotechnology which resistant coli
involves maintenance of sterile ambience is gene (2) Phosphodiester bonds
[NCERT Pg. 194] (3) Antibiotic Chromosomal Salmonella (3) Ionic bonds
(1) Genetic engineering sensitive DNA typhimurium
(4) Disulphide bonds
gene
(2) Bioprocess engineering
(4) Antibiotic Chromosomal Escherichia 10. The separated DNA fragments can be
(3) Developing a DNA vaccine visualised only after staining the DNA with
sensitive DNA coli
(4) Correcting a defective gene gene A and followed by exposure to B .
3. ______ gene codes for the proteins required 6. Restriction endonuclease and methylase Choose the option which correctly fill the
for the replication of plasmid were isolated from E.coli in the year blanks A and B respectively.
[NCERT Pg. 199] [NCERT Pg. 195]
[NCERT Pg. 198]
(1) ori (2) rop (1) 1972 (2) 1963
A B
(3) ampR (4) tetR (3) 1997 (4) 1990
(1) Ethidium bromide UV radiations
4. Genetic engineering is better over traditional 7. In Eco RI, the letter R is derived from
hybridization in [NCERT Pg. 194] [NCERT Pg. 195, 196] (2) Bromophenol blue UV radiations

(1) Introducing desirable genes only (1) Genus (2) Species (3) Ethidium bromide Visible light
(2) Introducing undesirable genes (3) Strain RY 13 (4) Strain Rd (4) Methylene blue Visible light
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11. The separated bands of DNA are cut out 14. Which vector is suitable to deliver desirable (1) Ca+2 (2) Na+
from the agarose gel and extracted from the genes into dicot plants? [NCERT Pg. 200] (3) K+ (4) Cl–
gel piece. This step is known as
(1) pBR322 18. Which of the following is incorrect w.r.t.
[NCERT Pg. 198]
(2) Disarmed Ti plasmid of Agrobacterium PCR? [NCERT Pg. 202, 203]
(1) Precipitation (2) Elution
(1) In vitro amplification of DNA
(3) Spooling (4) Fragmentation (3) Disarmed retrovirus
(2) Single set of RNA primers
12. If we ligate foreign DNA at Bam HI site of (4) Bacteriophage
pBR322, then the resultant recombinants (3) Primers are chemically synthesized
15. Complete the analogy w.r.t. DNA isolation
will show [NCERT Pg. 199] oligonucleotides
Bacteria : Lysozyme :: Fungus : ______
(a) Resistance to ampicillin (4) Thermostable DNA polymerase is used
[NCERT Pg. 201]
(b) Sensitivity to tetracycline 19. Select the correct sequence for the steps
(1) Cellulase (2) Chitinase involved in PCR [NCERT Pg. 202]
(c) Resistance to tetracycline
(3) Amylase (4) DNAse (1) Denaturation → Annealing → Extension
(d) Sensitivity to ampicillin
(1) a and b (2) a and c 16. All of the following are methods of direct (2) Annealing → Denaturation → Extension
(3) b and d (4) c and d gene transfer except [NCERT Pg. 201]
(3) Denaturation → Extension → Annealing
13. Recombinant host cells in which foreign (1) Micro-injection (4) Extension → Annealing → Denaturation
DNA is present in the coding sequence of (2) Biolistics 20. What is not present in the simple stirred-
enzyme β-galactosidase will produce
(3) Gene gun tank bioreactor? [NCERT Pg. 204]
[NCERT Pg. 200]
(4) Disarmed pathogen (1) Foam breaker
(1) Blue coloured colonies
(2) White coloured colonies 17. To make the host cells competent, they are (2) Temperature control system

(3) Pink coloured colonies treated with specified concentration of (3) Sampling port

(4) No colonies [NCERT Pg. 200] (4) Sparger

1. EFB stands for ______. [NCERT Pg. 193] 3. ______ sequence is responsible for 5. The cutting of DNA at specific locations
initiating replication in plasmid.
2. The two core techniques that enabled birth became possible with the discovery of
[NCERT Pg. 194]
of modern biotechnology are ______
4. First recombinant DNA was made by ______ enzymes, which are also called
engineering and ______ engineering. ______ and ______ in the year ______.
______. [NCERT Pg. 194, 195]
[NCERT Pg. 193, 194] [NCERT Pg. 194]
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6. The linking of antibiotic resistance gene with 10. DNA fragments are ______ charged 16. ______ cells are bombarded with high
the plasmid vector became possible with the molecules that move towards ______ during velocity micro-particles of gold or tungsten
enzyme ______. [NCERT Pg. 195] gel electrophoresis. [NCERT Pg. 198] coated with DNA in a method known as
11. DNA fragments separate according to their ______ or ______. [NCERT Pg. 201]
7. The first restriction endonuclease is ______.
______ through ______ effect provided by
Its recognition sequence is ______ long. 17. Since DNA is a ______ molecule, it cannot
the agarose gel. The ______ the fragment
[NCERT Pg. 195] pass through cell membranes.
size, the farther it moves. [NCERT Pg. 198]
8. First letter in the name of restriction 12. ______ sequence controls the copy number [NCERT Pg. 200]

endonuclease come from ______ and the of the linked DNA. [NCERT Pg. 199] 18. If any protein encoding gene is expressed in
second two letters come from the ______ of 13. Agrobacterium tumefaciens, a pathogen of a heterologous host, it is called a ______
the prokaryotic cell from which they were several ______ plants is able to deliver an [NCERT Pg. 203]
alien DNA through its ______ plasmid.
isolated. [NCERT Pg. 195] 19. Taq polymerase, a ______ DNA
[NCERT Pg. 200]
9. Restriction enzymes cut the strand of DNA polymerase, is isolated from ______.
14. Disarmed ______ are now commonly used
a little away from the centre of the [NCERT Pg. 203]
to deliver desirable genes into animal cells.
palindrome sites, but between the ______ 20. Separation and purification of desired
[NCERT Pg. 200]
on the opposite strands. This leaves single product in genetic engineering are included
15. In a method known as ______, recombinant
stranded portions on each strand called in ______ processing.
DNA is directly injected into the nucleus of
______. [NCERT Pg. 197] an animal cell. [NCERT Pg. 201] [NCERT Pg. 204, 205]

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