Biotechnology deals with techniques of using live

organisms or enzymes from organisms to produce

products and processes useful to humans. In this sense,
making curd, bread or wine, which are all microbe-
mediated processes, could also be thought as a form of
biotechnologr. However, it is used in a restricted sense
today, to refer to such of those processes which use
genetically modified organisms to achieve the same on a
larger scale. Further, many other processes/teéhniques
are also included under biotechnologr. For example, in
vitro fertilisation leading to a 'test-tube' baby,
synthesising a gene and using it, developing a DNA
vaccine or correcting a defective gene, are all part of
biotechnology.
The European Federation of Biotechnology
given a definition of biotechnologr that
encompasses both traditional view and modern
molecular biotechnology.
The definition given by EFB is as follows:
 The integration of natural science and organisms/
cells, parts thereof, and molecular
analogues for products and services'/

11.1 PRINCIPLES OF BIOfECHNOLOGY

Arnong many, the two core techniques that
enabled birth of modern biotechnology are :
(i) Genetic engineering : Techniques to alter the
chemistry of genetic material (DNA and
RNA),
 to introclucc these into organisms and thus change

phenotype of he host organisrn.

(ji) Bioprocess engineering: Maintenance of sterile (microbial

contamination -free) anil)icnce in chemical engineering
processes to enable growth of only thc desired
microbe/eukaryot ic cell in large quantities Cor the
manufacture of biotechnological products like antibiotics,
vaccines, enzymes, etc.
Let us now understand the conceptual development of the
principles of genetic engineering.
You probably appreciate the advantages of sexual reproduction
over asexual reproduction. The former provides opportunities for
variations and formulation of unique combinations of genetic setup,
some Of which may be beneficial to the organism as well as the
population. Asexual reproduction preserves the genetic information,
while sexual reproduction permits variation. Traditional
hybridisation procedures used in plant and animal breeding, very
often lead to inclusion and multiplication of undesirable genes along
with the desired genes. The techniques ofgenetic
engineering which include creation of recombinant DNA, use of
gene cloningånd gene transfer„overcome this limitation and allows
us to isolate and introduce only one or a set of desirable genes
without introducing undesirable genes into the target organism.
Do you know the likely fate of a piece of DNA, which is somehow
transferred into an alien organism? Most likely, this piece of DNA would
not be able to multiply itself in the progeny cells of the organism. But,
when it gets integrated into the genome of the recipient, it may multiply
and be inherited along with the host DNA. This is because the alien piece
of DNA has become part of a chromosome, which has the ability to
replicate. In a chromosome there is a specific DNA sequence called the
origin of replication, which is responsible for initiating replication.
Therefore, for the multiplication of any alien piece of DNA in an
organism it needs to be a part of a chromosome(s) which has a specific
sequence known as 'origin of replication'. Thus, an alien DNA is
linked with the origin of replication, so that, this alien piece of DNA
can replicate and multiply itselfin the host organism. This can also be
called as cloning or making multiple identical copies of any template
DNA.
Let us nowfocus on the first instance ofthe construction of an
artificial recombinant DNA molecule. The construction of the first
recombinant DNA emerged from the possibilityof linking a gene
encoding antibiotic resistance with a native plasmid (autonomously
replicating circular
extra-chromosomal DNA) of Salmonella typhimuriurn. Stanley Cohen
and
Herbert Boyert accomplished this in 1974 by isolating the antibiotic
resistance gene by cutting out a piece of DNA from a plasmid which
was responsible for confey@g antibiotic resistance. The cutting of
DNA at specific locations became possible with the discovery of the so-
called
'molecular scissors'— restriction enzymes. The cut piece of DNA
was then linked with the plasmid DNA. These plasmid DNA act as
vectors to transfer the piece of ONA attached to it. You probably
know that mosquito acts as an insect vector to transfer the malarial
parasite into human body. In the same way, a plasmid can be used as
vector to deliver an alien piece of DNA into the host organism. The
linking of antibiotic resistance gene with the plasmid vector became
possible with the enzyme DNA ligase, which acts on cut DNA
molecules and joins their ends. This makes a new combination of
circular autonomouslyyeplicating DNA created in vitro and is known
as recombinant DNA. When this DNA is transferred into Escherichia
coli, a bacterium closely related to Salmonella, it could replicate
using the new host's DNA polymerase enzyme and make multiple
copies. The ability to multiply copies of antibiotic resistance
gene in E. coli was called cloning of antibiotic resistance gene in E.
coli.
You can hence infer that there are three basic steps in genetically
modifying an organism
(i) identification of DNA with desirable genes;
(ii) introduction of the identified DNA into the host;
(iii) maintenance of introduced DNA in the host and transfer of the
DNA to its progeny.
11.2 Tools OF RECOMBINANT DNA TECHNOLOGY
Now we know from the foregoing discussion that genetic engineering or
recombinant DNA technology can be accomplished only if we have the
key tools i.e., restriction€ryymes, polymerase enzymes, ligases, vectors
and the host organism. Let us try to understand some of these in detail.

11.2.1 Restriction Enzymes on a

In the year 1 the two enzymes responsible for restricting the growth
of bacteriophage in Escherichia coliwere isolated. One of these
added ynethyl groups to DNA, while the other cut DNA. The later
was called restriction endonuclease.
The 'first restriction endonuclease—Hind Il/ whose functioning
depended on a specific DNA nucleotide sequence was isolated and
characterised five years later. It was found that Hind Il always cut
DNA molecules at a particular point by recognising a specific
sequence of 'Six base pairS. This specific base sequence is known as
the recognition sequence for Hind Il. Besides Hind Il, today we
know more than 900 restriction enzymes that have been isolated
from over 230 strains Ofbacteria each of which recognise different
recognition sequences.
Ihe convention for naming these enzymes is the first letter of the
name comes from the genus and the second two letters come from
the spgqies of
cell from which they were isolated. e.g. , EcoRI comes
from Escherichia coli RY 13. In EcoRI, the letter 'R' is derived from the
name of
 strain. Roman numbers following the names indicate the order in
the enzymes were isolated from that strain of bacteria.
Restriction enzymes belong to a larger class of enzymes called
nucleases. These are of two kinds; exonucleases and endonucleases
the DNA
whereas, endonucleases make cuts at specifiepositions within the
DNA.
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
backbones (Figure 11.1). Each restriction endonuclease recognises a
specific palindromic nucleotide sequences in the DNA.
Action of Restriction enzyme
The enzyme cuts both DNA EcoRI cuts the DNA between bases
strands at the same site G and A only when the sequence
GAA"ITC is present in the
DNA
Vector DNA Foreign DNA

DNA fragments join at sticky ends

Recombinant DNA

Figure 11.1 Steps in formation of recombinant DNA by action of restriction endonuclease

enzyme - EcoRI
 Do you know what palindromes are? These are groups of
letters that form the same words when read both forward and
backward . e.g.. "MALAYALAM". As against a word-palindrome
where the same word is read in both directions, the palindrome
in DNA is a sequence of base pairs that reads same on the two
strands when orientation Of
reading is kept the same. For example, the following sequences
reads the same on the two strands in 5' -5 3' direction. This is also
true if read in the 3' 5' direction.
5' —— GAATPC 3'
3' crrAAG
Restriction enzymes cut the strand of DNA a little away from the
centre of the palindrome sites, but between the same two bases on the
opposite strands. This leaves single stranded portions at the ends.
There are overhanging stretches called sticky ends on each strand
(Figure 11.1). These are named so because Chey form hydrogembonds
with their cornplementary cuc counterparCs.
This stickiness of the ends facilitates the action of the enzyme DNA
ligase.
Restriction endonucleases are used in genetic engineering to form
'recombinant' molecules of DNA. which are composed of DNA from
different sources/genomes.
When cut by the sarne restriction enzyme, the resultant DNA
fragments have the same kind of 'sticky-ends' and, these can be
joined together (end-to-end) using DNA ligases (Figure 11.2).
 Figure 11.2 Diagrammatic representation of recombinant DNA technologv

You may realiscd that normally, unless one cuts the the
DNA with the same restriction enzyme, the recombinant

Separation and isolation of DNA fragments : cutting of

endonucleases results in the fragments of DNA can bc
separated by a technique known as gel electrophoresis.
DNA fragments are negatively charged molecules they can be
separated by forcing them to move towards the anode under an electric
field through a medium/matnx. Nowadays the most commonly used
matrix is is a natural extracted from sea weeds. The DNA
fragments separate (resolve) according to their size through sieving
effect by the agarose gel. Hence, the smaller the fragment size, the
farther it moves. Ink at the Figure 11.3 and guess at which end of the
gel the sample was loaded
The separated DNA fragments can be
visualised only after staining the DNA
Wells with a compound known as
ethidium bromide Ilowed b exposure to IN
radiation (you cannot see pure DNA
fragments in the visible light and without
staining). You can see bright orange coloured
bands of DNA in a ethidium bromide stained
ge exposed to UV light (Figure 11.3). lhe
separated bands of DNA are cut out Figure I i .3 A typical agarose gel from the
agarose gel and extracted electrophoresis showing from the gel piece. This step is
known migration (lane l) and of digested undigestedset of as elution. The DNA
fragments DNA fragments (lane 2 to 4) purified in this way are used in
constructing recombinant DNA by joining them with cloning vectors.
11.2.2 Cloning Vectors
You know that plasmids and bacteriophages have the ability to
replicate within bacterial cells independent of the control of
chromosomal DNA Bacteriophages because of their high number er
cell, have very high

copy numbers of their genome within the bacterial cells. Some

plasmids

may have only one or two copies per cell whereas others may

15-100 copies per cell. Their numbers can go even

higher. If we are
 to link an alien piece of DNA with bacteriophage or plasmid DNA,
we cal multiply its numbers equal to the copy number of the plasmid
0 bacteriophage. are engineered in such a (hat they help easy linking
ollt•oreign DN,'N and selection recombinant from non-
recombinants..
 The following are the features that are required to facilitate cloning
into a vecton

(i) Origin of replication (ori) : This is a sequence from where

replication starts and any piece of DNA when linked to Chis
sequence can be made to replicate within the host cells. This
sequence is also e responsible for controlling the copy number
of the linked DNA. So,
if one wants to recover many copies of the target DNA it should be
cloned in a vector whose origin support high copy number.
(ii) Selectable marker : In addition to 'ori', the vector requires a
selectable marker, which helps in identifying and eliminating
nontransformants and selectively permitting the growth of the
transformants. Transformation is a procedure through which a
piece of DNA is introduced in a host bacterium (you will study
the process in subsequent section). 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.

(iii) Cloning sites: In order to link the EcoR1
alien DNA, the vector needs to have
Pvu 1
very few, preferably single, recognition
sites for the commonly Pst 1 BamH 1
used restriction enzymes. Presence of more
than one recognition sites within

11

Sal 1
 the vector will generate several fragments, which will complicate
the gene cloning (Figure 11.4). The ligation of alien DNA is carried
out at a restriction site present in one of the two antibiotic resistance
genes. For

example, you can ligate a foreign DNA Figure 11.4 E. coli cloning vector pBR322
at the BamH I site of tetracycline showing restriction sites (Hind 111,
resistance gene in the vector EcoR1, Bar-nH 1, Sal 1, Pvu 11, Pst
1, Cia 1), ori and antibiotic resistance
pBR322. The recombinant plasmids genes (ampR and tetR). rop codes for
will lose tetracycline resistance due the proteins involved in the
to insertion of foreign DNA but can replication of the plasmid.
still be selected out from non-
recombinant ones by
plating the transformants on

tetracycline containing medium. The transformants growing on

ampicillin containing medium are then transferred on a medium
containing tetracycline. The recombinants will grow in ampicillin
containing medium but not on that containing tetracycline. But,
non- recombinants will grow on the medium containing both the
antibiotics. In this case, one antibiotic resistance gene helps in
selecting the transformants, whereas the other antibiotic resistance
 doc to insert ion' of alien DNA, and helps in
'sclcct ion of rccorrjbinarjts,

Selection of duc to inactivation of antibiotics is a

cornbcr»omc procedurc becausc it requires simultaneous
plating
on two plates having different antibiotics. njcrefore. alternative
selcefablc markers have becn dcvclopcd which differentiate
recombinants from non-recombinants on the basis of their ability
to produce colour in the presence of a chromogenic substrate. In
thjb, a recombinant DNA is inserted within the coding sequence
of an This results into inactivation of
the gene for synthesis of this enzyrne, which is referred to as
insertional inactivation. The presence of a chromogenic substrate
gives blue coloured colonies if the plasmid in the bacteria does
not have an insert. Presence of incert results into insertional
inactivation of the
-galactosidase and the colonies do not produce any colour, these
arc jdentjfied as recombinant colonies.
(jvj Vectorsfor cloning genes in plants and animaLs : You may be
surprised to know that we have learnt the lesson oftransferring genes
jnto plants and animals from bacteria and viruses which have known
this for ages how to deliver genes to transforrn eukaryotic cells and
force them to do what the bacteria or viruses want. For example,
Vigrobarterinrn turrüfaciens, a pathogen of several dicot plants is
able to dcljvcr a piece of DNA known as 'T -DNA' to transform
normal plant cells into a tumor and direct these tumor cells to
produce the chemicals required by the pathogen. Similarly.
retroviruses in anim have the abjljty to transform normal cells into
cancerous cells. A better understanding of the art of delivering genes
by pathogens in their eukaryotic hosts has generated knowledge to
transform these tools of pathogens into useful vectors for delivering
genes of interest to humans. tumor inducing (Ti) plasmid of
Agrobacterium turn(facierus has now been modified into a cloning
vector which is no more pathogenic to the plants but is still able to
use the mechanisms to deliver genes of our interest into a variety of
plants. Similarly, retroviruses have also been disarmed and are now
used to deliver desirable genes into animal cells. So, once a gene or a
DNA fragment has been ligated into a suitable vector it is transferred
into a bacterial, plant or animal host (where it multiplies).

11.2.3 Competent Host (For Transformation with

Recombinant DNA)
 Since ONA is a hydrophilic molecule, it cannot pass through c ell
membranes, Why? In order to force bacteria to take up the plasmid,
the bacterial cells must first be made 'competent' to take up DNA. Thi s
is
done by treating them with a specific concentration of a divalent
cation such as calcium; which increases the efficiency with which
DNA enters
 bacterium through pores in its cell wall, I)NA
can then be forced into such cells by incubating the cells with
recombinant JONA on ice. followed by placing Chem briefly at
420C (heal shock), and then putting them back on ice. This enables
the bacteria to lake up the rcombinant DNA.
Illis is not the only way to introduce alien DNA into host cells.
In a method known as recombinant DNA is
directly injected into the nucleus of an animal cell. In another
method, suitable for plants, cells are bombarded with high
velocitymicro-partic!es ofgold or tungsten coated with DNA in a
method known as biolistics or gene gun. And the
last method uses vectors, which
when allowed to infect the cell, transfer the recombinant DNA into
the host.
Now that we have learnt about the tools for constructing recombinant
DNA let us discuss the processes facilitating recombinant DNA

Recombinant DNA technology involves several steps in

specific sequence such as isolation of DNA, fragmentation
of DNA by restriction endonucleases, isolation of a desired
DNA fragment, ligation of the DNA fragment into a vector,
transferring the recombinant DNA into the host, culturing
the host cells in a medium at large scale and extraction of
the desired product. Let us examine each of these steps in
some details.

11.3.1 Isolation of the Genetic Material (DNA)

Recall that nucleic acid is the genetic material of all
organisms without exception. In majority of organisms this
is deoxyribonucleic acid or DNA. In order to cut the DNA
with restriction enzymes, it needs to be in pure form, free
from other macro-molecules. Since the DNA is enclosed
within the membranes, we have to break the cell open to
release DNA along with other macromolecules such as
RNA, proteins, polysaccharides and also lipids. This can be
achieved by treating
the bacterial cells/plant or animal tissue with enzymes such Figure 11.5 DNA that
as lysozyme (bacteria), cellulase (plant cells), chitinase separates out can be
(fungus). removed by spooling
You know that geoes are located on long molecules of DNA inter-
wined with proteins such as histones. The RNA can be removed by
treatment with ribonuclease whereas proteins can be removed by
treatment with protease. Other molecules can be removed by
appropriate treatments and purified DNA ultimately precipitates the
addition

Ofehilled ethanol'This can be seen as collection of fine threads in the

suspension (Figure 11.5).
 11.3.2 Cutting of DNA at Specific Locations
Restriction enzyme digestions are performed by incubating purified
molecules with the restriction enzyme, at the optimal conditions
for that specific enzyme. Agarose gel electrophoresis is employed to
check the progression of a restriction enzyme digestion. DNA is a
negatively charged molecule, hence it moves towards the positive
electrode (anode) (Figure 11.3). The process is repeated with the
vector DNA also.
The joining of DNA involves several processes. After having cut
the source DNA as well as the vector DNA with a specific restriction
enzyme, the cut out 'gene of interest' from the source DNA and the
cut vector with space are mixed and ligase is added. This results in
the preparation of recombinant DNA.

11.3.3 Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction. In this reaction, multiple
copies of the gene (or DNA) of interest is synthesised in vitro using
two
Region to be amplified

3'
ds DNA
5'
Denaturation
3'
Primers Annealing

5'

Extension

Amplified
billion times)

11.6 Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation; (if) Primer
annealing; and (iii) Extension of primers
 sets of primers (small to tile regions cheniically of DNA) synthesisccl and the
enzyjnc oligonucleot DNA ides polymerasethat arc

enzynme extends the prinners using the nucleotides provided in

Che reaction and the <enotnic DNA as template. If the process of
replication of DNA is repeated many times, the segment of DNA can
be amplified to approximately billion times, i.e., 1 billion copies are
made. Such repeated amplification is achieved by the use of a
thermostable DNA polymerase (isolated from a bacterium, Thermus
aquaticus), which remain active during the high temperature induced
denaturation of double stranded DNA. The amplified fragment if
desired can now be used to ligate with a vector for further cloning
(Figurel 1.6).
11.3.4 Insertion of Recombinant DNA into the Host
Cell/Organism
There are several methods of introducing the ligated DNA into recipient cells.
Recipient cells after making them 'competent' to receive, take up DNA
present in its surrounding. So, if a recombinant DNA bearing gene for
resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the
host cells become transformed into ampicillin-resistant cells. If we spread the
transformed cells on agar plates containing ampicillin, only
transformants will grow, untransformed recipient cells will die. Since, due to
ampicillin resistance gene, one is able to select a transformed cell in the
presence of ampicillin. Ihe ampicillin resistance gene in this case is called a
selectable marker.

11.3.5 Obtaining the Foreign Gene Product

When you insert a piece of alien DNA into a cloning vector and transfer it
into a bacterial, plant or animal cell, the alien DNA gets multiplied. In
almost all recombinant technologies, the ultimate aim is to produce a
desirable protein. Hence, there is a need for the recombinant DNA to be
expressed. The foreign gene gets expressed under appropriate conditions.

The expression of foreign genes in host cells involve understanding many

technical details.
After having cloned the gene of interest and having optimised
the conditions to induce the expression of the target protein, one
has to consider producing it on a large scale. Can you think of any
reason why there is a needfor large-scale production? If any protein
encoding genets expressed in a heterologous host, it is called a
recombinant PtOtein.lThe cells harbouring cloned genes of interest
may be grown On a small scale in the laboratory. The cultures may
be used for extracting the desired protein and then purifying it by
using different separation techniques. cells can also be
multiplied in a continuous culture system wherein the 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. This type of culturing method produces
larger biomass leading to higher yields of desired protein.
Small volume cultures cannot yield appreciable quantities of products
To produce in large quantities, the development of bioreactors, where
large volumes ( 1 OO- 1000 litres) of culture can be processed, was
required Thus, 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. A bioreactor
provides the optimal conditions for achieving the desired product by
providing optimum growth conditions (temperature, pH, substrate, salts,
Vitamins, p»gen).
The most commonly used bioreactors are of stirring type, which
are shown in Figure 11.7.

(a) (b)

gure 11.7 (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which
sterile air bubbles are sparged

A stirred-tank reactor is usually cylindrical or with a curved base

tg facilitate the mixing of the reactor contents. The stirrer facilitates
even mixing and oxygen availability throughout the bioreactor.
Alternatively air can be bub led through the reactor. If you look at
the figure closely you will see that 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.
11.3.6 Downstream Processing
After completion of the biosynthetic stage, the product has to be
subjected through a series ofprocesses before it is ready for marketing
as a finished
 roduct. 'Ille processes include separation and purificat ion, which are collectively
referred to as downstream processing. product has to be formulated with suitable
preservatives. Such formulation has to undgcgo thorou h clinical trials as in caseof
drugs. Strict quality control testing for each product is also required. Il-me downstream
processing and quality control testing vary from product to product.