CHAPTER: 11

BIOTECHNOLOGY
PRINCIPLES AND PROCESS
Biotechnology: It is a branch of
biology which deals with techniques
used in living organism particularly in
the cells for betterment. E.g.
production of genetically modified
food (G.M. Food), in vitro
fertilisation for test tube baby,
development of DNA vaccine etc.
Principles
of biotechnology:
Genetic engineering: It is a technique
that changes the chemistry of genetic
material. Further this changed DNA or
RNA i.e. genetic material is introduced
into the host organism to change the
morphology.
Production of biotechnological
products: Various products like
vaccines, enzymes, antibiotics etc. are
prepared with the help of this technique.
What does genetic engineering
includes?
Creation of recombinant DNA.
Use of gene cloning.
Gene transfer.
Origin of replication: In a
chromosome there is a specific DNA
sequence which is responsible for
initiating the replication is called origin of
replication
Cloning of DNA: When any foreign
(Alien) DNA is linked with the ‘origin of
replication’, so that this foreign DNA can
replicate and multiply itself in the host
organism. This is called cloning or making
multiple identical copies of any template
of DNA.
 Construction of the first
recombinant DNA molecule:
Stanley Cohen and Herbert Boyer in
the year 1972, isolated an antibiotic
resistant gene from a plasmid
(autonomously replicating extra
chromosomal DNA) of the bacterium
Salmonella typhimurium.
Both isolated the antibiotic
resistance gene by cutting the
desired piece of DNA from plasmid.
 Cutting of a piece of DNA from a plasmid
was done with the help of restriction
enzymes, popularly known as molecular
scissors.
 The piece of DNA cut from the plasmid
was then linked with the plasmid DNA
which acts as vector. The linking of the
fragment of DNA with the vector was
done with the help of another enzyme
called DNA ligase.
 This newly formed DNA having integrated
fragment of antibiotic resistant gene is
called recombinant DNA.
The recombinant DNA is transferred
to bacterium E. Coli
 (Escherichia coli).This transfer of
recombinant DNA is exactly similar
to the transfer of malarial parasite
from diseased person into the
healthy human body through
mosquito.
The vector acts as a mosquito in
transferring the piece of DNA from
donor to the recipient organism.
Once the newly formed recombinant
DNA was transferred to the
bacterium E.coli, it replicates in the
host cell by using the enzyme DNA
polymerase.
It results in the formation of several
copies of recombinant DNA having
capability of antibiotic resistance.
Steps in genetically modifying
an organism:
Identification of DNA with
desirable genes.
Introduction of the identified DNA
into the host.
Maintenance of introduced DNA
into the host and transfer of the
DNA to its progeny.
Tools of recombinant DNA
technology: Biological tools for
recombinant DNA technology are:
Enzymes (restriction enzymes,
ligases, polymerase enzyme)
Vehicle or Vector DNA
Passenger DNA (Host organism)
Restriction enzyme: It belongs to a
class of enzymes called nucleases. They are of
two types:
 Exonucleases: Which cut off nucleotides from
5’ or3’ terminal ends of DNA molecule.
 Endonucleases: This cut DNA at any point
except the terminal ends.
 Stewart Linn and Werner Arber (1963) isolated
two enzymes from E.coli that were responsible
for restricting the growth of bacteriophage,
one of them added methyl groups to the DNA
and other cut the DNA into segments called
restriction endo nuclease.
 H.O. Smith, K.W.Wikox and T.J.Kelley (1968)
isolated and named the first restriction
endonuclease from haemophilus influenza
bacterium and called HindII. It always cut
the DNA molecule at a particular point by
recognising a specific sequence of six base
pairs called recognition sequence.
 The recognition sequence is a palindrome,
where the sequence of base pair reads the
same on both the DNA strands, when the
orientation of reading is kept the same. E.g.
5’- GAATTC-3’ and 3-’CTTAAG-5’.
 Each restriction endonuclease functions by
‘inspecting’ the length of a DNA sequence.
Once it finds its specific recognition sequence,
it will bind to the DNA and cut each of the two
strands of the double helix at specific points in
their sugar-phosphate backbone.
 As a result, single stranded portions called
sticky ends are produced at the end of the
DNA; this stickiness of the ends facilitates
the action of enzyme DNA ligase.
 When cut by the same restriction
endonuclease the DNA fragments yield the
same kind of sticky ends which can be joined
end to end by DNA ligase.
Nomenclature of restriction
enzyme:
 The name of enzyme is derived from the
name of the prokaryotic cell from which
the enzyme is isolated.
 The first letter of the genus becomes the
first letter of the name of enzyme. It is
written in capital letter.
 The first two letters of the species make
second and third letter of the name of
the enzyme. They are written in small
letter.
 All these letters are written in italics.
 The fourth letter of the enzyme is the first
letter of the strain. It is written in capital.
 The roman number written at the end of the
name indicates the order in which the enzyme
was isolated from the prokaryotic cell.
 For example EcoRI, isolated from Escherichia
coli RY13. It is named as follows:
 The capital letter E comes from Escherichia.

 The letters co comes from coli.

 The letter R comes from RY13 (strain)

 The roman number I indicates it was the first

enzyme isolated from the bacterium E.coli RY13.
Separation and isolation of DNA
fragments:
 The cutting of DNA by restriction
endonuclease results in the fragments of
DNA. These fragments can be separated by
a technique known as gel electrophoresis.
 DNA fragments are –vely charged
molecules, so separated by forcing them to
moves towards the anode under an electric
field through a medium/matrix. (Agarose- a
natural polymer extracted from sea weeds).
 The DNA fragments separate according to their
size through sieving effect provided by the
agarose gel. Thus, smallest fragments move
farther.
 The separated DNA fragments can be seen only
by staining the DNA with ethidium bromide
followed by exposure to UV radiations as orange
coloured bands of DNA.
 The separated bands are cut out from the
agarose gel and extracted from the gel piece.
This is called elution.
 The DNA fragments purified is thus, used in
constructing recombinant DNA by joining them
with cloning vectors.
Cloning vectors: Plasmids and
bacteriophage are commonly used vectors.
The following features are required to
facilitate cloning into a vector.
Ori (Origin of replication): This is a
sequence of base pair on DNA where
replication starts. Any piece of DNA
linked to this sequence can be made to
replicate within the host cells. This
sequence is also responsible for
controlling the copy number of the linked
DNA.
Selectable marker: In addition to ‘Ori’,
the cloning vectors require the presence of
selectable marker to identify and eliminate non-
transformants. On the other hand, it should
selectively permit the growth of the
transformants. Plasmids are characterised to
possess some important genes like the antibiotic
resistance gene, genes for production of toxic
substances, genes for tumour formation and
genes for nitrogen fixation etc. These genes may
act as selectable marker in transformations. For
e.g. the genes encoding resistance to antibiotics
such as ampicillin, chloramphenicol, tetracycline
etc. are the useful selectable marker for E.coli.
Cloning sites:
 The vector should have a few or at least one
recognition site, to like the foreign DNA.
 Presence of a particular recognition site enables
the particular restriction enzyme to cut the
vector.
 If a foreign DNA is ligated at BamHI site of
tetracycline-resistance gene in the vector
pBR322, the recombinant plasmid loses the
tetracycline resistance.
 It can still be selected out from the non-
recombinant ones by plating the transformants
on ampicillin containing medium.
 Those transformants which grow on ampicillin-
containing medium are then transferred to a
medium containing tetracycline.
 The recombinant will grow on ampicillin-
containing medium, but not on tetracycline-
containing medium, but non-recombinants will
grow on the both the media.
 In this case, one antibiotic gene helps in
selecting the transformants whereas the other
antibiotic-resistance gene gets inactivated and
helps in selection of recombinants.
 Another method to differentiate between
recombinants and non-recombinants is on the
basis of their ability to produce colour.
 In this method a recombinant DNA is
inserted within the coding sequence of
an enzyme β galactosidase.
 This results into inactivation of the
enzyme, which is called insertional
inactivation.
 The presence of a chromogenic
substrate gives blue coloured colonies if
the plasmid in the bacteria does not
have an insert where as recombinant
colonies do not produce any colour.
Vector for cloning genes in
plants and animals: A soil-inhabiting
plant pathogenic bacterium, Agrobacterium
tumefaciens, infects broad-leaved crops
including tomato, soyabean, sunflower and
cotton, but not the cereals. It causes
tumours called crown galls. Tumour
formation is induced by its plasmid, which
is therefore called Ti plasmid. The Ti
plasmid integrates a segment of its DNA,
termed T DNA, into the chromosomal DNA
of its host plant cell.
The T DNA causes tumours. As gene
transfer occurs without human effort,
the bacterium is known as ‘natural
genetic engineer’ of plants. Plant
molecular biologists have started using
Ti plasmids as vectors to transfer
foreign genes into the target plant
cells. They use a version of the plasmid
from which tumour- forming gene has
been eliminated. The transformed
bacteria do not cause disease.
  Competent Host (for
transformation with recombinant
DNA): DNA is a hydrophilic
molecule and cannot pass directly
through cell membrane. Therefore
certain treatments are made in host
cells so that they become competent
to take up DNA. There are different
methods:
Chemical methods: This is done by
treating host with a specific
concentration of a divalent cation, such as
Calcium, which increases the efficiency
with which DNA enters the bacterium
through pores in its cell wall. Recombinant
DNA can then be forced into such cells by
incubating the cells with recombinant
DNA on ice, followed by placing them
briefly at 420c, and then putting them
back on ice. This enables the bacteria to
take up the recombinant DNA.
Micro injection: In this method
recombinant DNA is directly injected into
the nucleus of an animal cell.
Biolistics or gene gun: This method
is suitable for plants. In this case cells are
bombarded with high velocity micro-
particles of gold or tungsten coated with
DNA.
Disarmed pathogen vectors:
Which when allowed infecting the cell,
transferring the recombinant DNA into
the host.
 Processes of recombinant DNA
technology: It involves the following steps:
Isolation of the genetic material
(DNA):
 Bacterial cells/plant or animal tissues are treated with
certain enzymes to break open the cell envelopes.
Bacterial cells are treated with lysozyme to dissolve the
bacterial cell walls. Plants cells are treated with enzyme
cellulase to dissolve cellulose cell wall and the enzyme
chitinase is used to dissolve fungal cell wall. As a result
of which DNA is released along with several other
macromolecules and impurities such as RNA, proteins,
polysaccharides and lipids.
 Next step is purification and separation
of DNA. The eukaryotic DNA molecules
are interwined with proteins such as
histones. These proteins can be removed
by treatment with enzyme protease. The
enzyme protease converts proteins into
amino acids. RNA is removed by treating
with enzyme ribonuclease.
 Finally pure DNA molecules are
precipitated out by adding chilled ethanol
and collected in the suspension.
Cutting of DNA at specific location:
Isolated and purified DNA molecules
are cleaved by using the enzyme
restriction endonuclease. The purified
and cleaved DNA molecules i.e. both
the source DNA and vector DNA are
mixed, in presence of enzyme ligases
so that their sticky ends join
resulting in the formation of
recombinant DNA.
Amplification of gene of interest
using PCR (Polymerase Chain
Reaction): In PCR multiple copies of the
gene (orDNA) of interest are made in vitro
using two sets of primers and enzyme DNA
polymerase. The enzyme extends the primers
using the nucleotides provided in the reaction
and the genomic DNA as template. If the
process of replication of DNA is repeated many
times then 1 billion copies are made. It can be
achieved by the use of a thermostable DNA
polymerase which remains active during the high
temperature induced denaturation of double
stranded DNA.
Insertion of recombinant DNA into
the host cell/ organism:
 Several techniques are used to introduce the
ligated DNA into recipient cells. Once a cell
receives a foreign DNA fragment and becomes
transformed into a genetically modified cell, it
starts behaving differently. For example, if a
rDNA bearing gene, which is resistant to antibiotic
ampicillin, is inserted in E.coli cells, the bacteria
become resistant to ampicillin. If such bacteria are
transferred on a culture plate containing the
antibiotic ampicillin only the resistant forms will
grow and other will die. Such ampicillin resistance
gene here is called a selectable marker.
 Obtaining the foreign gene product: The
recombinant gene is expressed in the form of
proteins. The transgenic cells may be cultured in
the laboratory to obtain the transgene product
on a small scale. The cultures may be used for
extracting the desired protein and then
purifying it by using different separation
techniques. The cells can also be multiplied in a
continuous culture system where in the used
medium is drained out from one side while the
fresh medium is added from the other to
maintain the cells in their physiologically most
active exponential phase. This type of culturing
method produces a larger biomass leading to
higher yield of desired protein.
Down streaming processing: The
foreign gene product obtained is
subjected to a series of process
before it is ready for marketing as a
finished product. The processes
include separation and purification
which is called downstream processing.
The product has to be formulated
with suitable preservatives and has to
undergo clinical trials. Strict quality
control testing is done.
Bioreactor: Bioreactors can be thought of
as vessels, in which raw materials are biologically
converted into specific products, individual
enzymes etc. using microbial plant, animal or
human cells. It provides the optimal conditions
for achieving the desired product by providing
optimum growth conditions (temperature, pH,
substrate, salts, vitamins, oxygen). The most
commonly used bioreactors are of stirring type.
A stirred tank bioreactor is usually a cylindrical
vessel or vessel with a curved base to facilitate
mixing of the contents. The stirrer present
facilitates the mixing and oxygen availability
throughout the bioreactor.