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BIOLOGY

gene gets ‘inactivated due to insertion’ of alien DNA, and helps in

selection of recombinants.

Selection of recombinants due to inactivation of antibiotics is a

cumbersome procedure because it requires simultaneous plating

on two plates having different antibiotics. Therefore, alternative

selectable markers have been developed which differentiate

recombinants from non-recombinants on the basis of their ability

to produce colour in the presence of a chromogenic substrate. In

this, a recombinant DNA is inserted within the coding sequence of

an enzyme, b-galactosidase. This results into inactivation of the

enzyme, 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

insert results into insertional inactivation of the â-galactosidase and

the colonies do not produce any colour, these are identified as

recombinant colonies.

(iv) Vectors for cloning genes in plants and animals : You may be

surprised to know that we have learnt the lesson of transferring genes

into plants and animals from bacteria and viruses which have known

this for ages – how to deliver genes to transform eukaryotic cells and

force them to do what the bacteria or viruses want. For example,

Agrobacterium tumifaciens, a pathogen of several dicot plants is able

to deliver 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 animals

have the ability 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. The tumor inducing (Ti) plasmid of Agrobacterium

tumifaciens 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 DNA is a hydrophilic molecule, it cannot pass through cell

membranes. Why? In order to force bacteria to take up the plasmid, the

bacterial cells must first be made ‘competent’ to take up DNA. This is

done by treating them with a specific concentration of a divalent cation,

such as calcium, which increases the efficiency with which DNA enters

2015-16

BIOLOGY

gene gets ‘inactivated due to insertion’ of alien DNA, and helps i

selection of recombinants.

Selection of recombinants due to inactivation of antibiotics is a

cumbersome procedure because it requires simultaneous platin

on two plates having different

antibiotics. Therefor

alternativ

selectable markers have been developed which differentia

recombinants from non-recombinants on the basis of their ability

roduce colour in the

resence of a chrom

enic substrate.

this, a recombinant DNA is inserted within the coding sequence of

n of t

. Th

lonies if

sence of

dase a

fied a

may b

kno

ells a

is ab

duce t

anima

cells. A

gens i

m thes

nterest

acterium

ch is

hanism

imilarly,

deliver

agment

acterial,

Since DNA is a hydrophilic molecule, it cannot pass through ce

membranes.

? In order to force bacteria to take up the plasmid, t

bacterial cells must first be made ‘competent’ to take up DNA. This

done by treating them with a specific concentration of a divalent cation,

such as calcium, which increases the efficienc

with which DNA enter

2015-1

220000

this, a recombinant DNA is inserted within the codin

an enzyme,

-galactosidase. This results into inactivatio

me, which is referred to as

insertional inactivati

presence of a chromogenic substrate gives blue coloured co

the plasmid in the bacteria does not have an insert. Pre

insert results into insertional inactivation of the

-g

alactosi

the colonies do not pr

oduce any colo

, these ar

e identi

recombinant colonies.

(iv)

ectors for

loni

enes i

lants an

nimals :

Y Y

rised to know that we have learnt the lesson of transferri

into plants and animals from bacteria and viruses which have

this for ages – how to deliver genes to transform eukaryotic c

force them to do what the bacteria or viruses want. For ex

Agrobacterium tumifaciens

, a pathogen of several dicot plants

to deliver a piece of DNA known as

‘

-DNA’ to transfor

plant cells into

tumor

and direct these tumor cells to pro

chemicals required by the pathogen. Similarly, retroviruses in

have the ability to transform normal cells into

cancerous

better understanding of the art of delivering genes by patho

their eukaryotic hosts has generated knowledge to transfor

tools of pathogens into useful vectors for delivering genes of i

to humans. The tumor inducing (T

i) plasmid of

Agr

tumifaciens

has now been modified into a cloning vector whi

more patho

nic to the plants but is still able to use the mec

to deliver genes of our interest into a variety of plants. S

retroviruses have also been disarmed and are now used to

desirable genes into animal cells. So, once a gene or a DNA fr

has been l

ated into a suitable vector it is transferred into a b

ant or animal host (where it multi

ies).

11.2.3

Competent Host (For Transformation with

Recombinant DNA)

Si DNA is hyd philic ol ul it ot th

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

201

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 42

C (heat shock), and then

putting them back on ice. This enables the bacteria to take up the

recombinant DNA.

This is not the only way to introduce alien DNA into host cells. In a

method known as micro-injection, recombinant DNA is directly injected

into the nucleus of an animal cell. In another method, suitable for plants,

cells are bombarded with high velocity micro-particles of gold or tungsten

coated with DNA in a method known as biolistics or gene gun. And the

last method uses ‘disarmed pathogen’ 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 technology.

11.3 PROCESSES OF RECOMBINANT DNA TECHNOLOGY

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 as

lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).

You know that genes are located on long molecules of DNA

interwined 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 out after the addition

of chilled ethanol. This can be seen as collection of fine threads in the

suspension (Figure 11.5).

Figure 11.5 DNA that

separates out can be

removed by spooling

2015-16

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

0011

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 b

placin

them briefly at 42

C (heat shock), and then

putting them back on ice. This enables the bacteria to take up the

recombinant DNA.

This is not the only way to introduce alien DNA into host cells. In a

method known as

micro-injectio

, recombinant DNA is directly injected

into the nucleus of an animal cell. In another method, suitable for plants,

cells are bombarded with high velocity micro-particles of gold or tungsten

coat

last

infe

DNA,

Reco

sequ

rest

liga

reco

medi

Let

11.3

Reca

with

deox

rest

macr

memb

with

poly

the

inte

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 out after the addition

of chilled ethanol. This can be seen as collection of fine threads in the

suspension (Figure 11.5).

2015-1

lls are bombarded with h

h veloci

micro-particles of gold or tungsten

ated with DNA in a method known as

biolistics

gene gun

. And the

st method uses ‘disarmed pathogen’ vectors, which when allowed to

fect the cel

transfer the recombinant DNA into the host.

Now that we have learnt about the tools for constructing recombinant

A, let us discuss the processes facilitating recombinant DNA technology.

1.3 P

ROCESSES

ECOMBINANT

DNA T

ECHNOLOGY

combinant DNA technology involves several steps in specific

quence such as isolation of DNA, fragmentation of DNA by

striction endonucleases, isolation of a desired DNA fragment,

gation of the DNA fragment into a vector

, transferring the

combinant DNA into the host, culturing the host cells in a

dium at large scale and extraction of the desired product.

us examine each of these steps in some details.

.3.1 Isolation of the Geneti

rial

(

DNDN

A)A)

call that nucleic acid is the genetic material of all organisms

thout exception. In majority of organisms this is

oxyribonucleic acid or DNA. In order to cut the DNA with

striction enzymes, it needs to be in pure form, free from other

macro-molecules. Since the DNA is enclosed within the

mbranes, we have to break the cell open to release DNA along

th other macromolecules such as RNA, proteins,

lysaccharides and also lipids. This can be achieved by treating

bacterial cells/

ant or animal tissue with enzymes such as

(bacteria),

lant cells),

(fu

us).

ou know that genes a

e located on long molecules of DNA

terwined with

oteins such as histones. The RNA can be removed

eatment with ribonuclease whereas proteins can be removed by

Figure 11.5

separates out can be

removed by spooling

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BIOLOGY

11.3.2 Cutting of DNA at Specific Locations

Restriction enzyme digestions are performed by incubating purified DNA

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

Figure 11.6 Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation;

(ii) Primer annealing; and (iii) Extension of primers

202

2015-16

BIOLOGY

ng of DNA at Specific Locations

Restrictio

n enzyme digestions are performed by incubating purified DNA

molecules with the restriction enz

e, at the

timal conditions for that

specific enzyme. Agarose gel electrophoresis is employed to check th

progression of a restriction enzyme digestion. DNA is a negatively charg

molecule, hence it moves towards the positive electrode (anode)

(Figure 11.3). The process is repeated with the vector DNA also.

The

inin

of DNA involves several

ocesses. After havin

cut th

nzyme,

tor wi

ration of

multip

ng t

Figure 11.6

Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation;

(ii) Primer annealing; and (iii) Extension of primers

2015-1

source DNA as well as the vector DNA with a specific restriction e

the cut out ‘gene of interest’ from the source DNA and the cut vec

space are mixed and ligase is added. This results in the prepa

recombinant DN

11.3.3 Amplification of Gene of Interest usi

PCR stands for

Polymerase Chain Reaction

In this reaction,

copies of the gene (or DNA) of interest is synthesised

usi

220022

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

203

sets of primers (small chemically synthesised oligonucleotides that are

complementary to the regions of DNA) and the 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, 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 fr

om 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 (Figure11.6).

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. The 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 need for large-scale production? If any protein encoding

gene is expressed in a heterologous host, it is called a recombinant

protein. The 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.

The 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

2015-16

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

0033

sets of primers (small chemically synthesised oligonucleotides that are

lementar

to the r

ions of DNA) and the enz

e DNA

merase.

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, the segment of DNA can be amplified

to approximately billion times, i.e., 1 billion copies are made. Such

ated a

lification is achieved b

the use of a thermostable DNA

polymerase (isolated fr

om a bacterium

Ther

mus aquaticus

), which

remain active during the high temperature induced denaturation of

doub

used

11.3

Ther

cells.

DNA

for re

cells,

we s

tran

to a

pres

11.3

When

into

almo

desi

expr

The

cond

cons

why

gene

extracting the desired protein and then purifying it by using different

separation techniques.

The 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

iolo

call

most

2015-1

main active during the high temperature induced denaturation of

uble stranded DNA. The amplified fragment if desired can now be

ed to ligate with a vector for further cloning (Figure11.6).

DNA i

Cell/Organism

ere are several methods of introducing the ligated DNA into recipient

lls. Recipient cells after making them ‘competent’ to receive, take up

DNA present in its surrounding. So, if a recombinant DNA bearing gene

resistance to an antibiotic (e.g., ampicillin) is transferred in

E. coli

lls, the host cells become transformed into am

cillin-resistant cells. If

spread the transformed cells on agar plates containing ampicillin, only

ansformants will grow, untransformed recipient cells will die. Since, due

ampicillin resistance gene, one is able to select a transformed cell in the

esence of ampicillin. The ampicillin resistance gene in this case is called

selectable marke

.3.5 Obtaining the Foreign

G G

enen

Prod

When you insert a piece of alien DNA into a cloning vector and transfer it

to a bacterial, plant or animal cell, the alien DNA

gets multiplied. In

most all recombinant technologies, the ultimate aim is to produce a

sirable protein. Hence, the

re is a need for the recombinant DNA to be

pressed. The foreign gene gets expressed under appropriate conditions.

e expression of foreign genes in host cells involve understanding many

After having cloned the gene of interest and having optimised the

nditions to induce the expression of the target protein, one has to

nsider

roduci

it on a lar

scale.

Can

u think

reason

why there is a need

r large-scale production

? If any protein encoding

ne is expressed in a heterologous host, it is called a

recombinant

otein

. The cells harbouri

cloned

nes of interest ma

rown

a small scale in the laboratory. The cultures may be used for

tracting the desired protein and then purifying it by using different

204

BIOLOGY

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

facilitate the mixing of the reactor contents. The stirrer facilitates even

mixing and oxygen availability throughout the bioreactor. Alternatively

air can be bubbled 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 of processes before it is ready for marketing as a finished

active log/exponential phase. This type of culturing method produces a

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 (100-1000 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,

oxygen).

The most commonly used bioreactors are of stirring type, which are

shown in Figure 11.7.

Figure 11.7 (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which

sterile air bubbles are sparged

(a)

(b)

2015-16

BIOLOGY

base

es eve

natively

closely

delivery

em,

cultu

can be withdrawn

eriodicall

11.3.6 Downstream Processing

After co

mpletion of the biosynthetic stage, the product has to be subjecte

through a series of processes before it is ready for marketing as a finishe

active log/exponential phase. This type of culturing method produces a

larger biomass leading to higher yields of desired

protei

Small volume cultures cannot

ield a

reciable

uantities of

roducts.

To produce in large quantities, the development of

bioreactors

, wher

large volumes (100-1000 litres) of culture can be processed, was required.

Thus, bioreactors can be thought of as vessels in which raw materials ar

biologically converted into specific products, individual enzymes, etc.,

using microbial plant, animal or human cells. A bioreactor provides th

timal conditions for achievi

the desired roduct b ovidin

tamins,

hich a

which

2015-1

220044

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

facilitate the mixing of the reactor contents. The stirrer facilitat

mixing and oxygen availability thr

oughout the bi

eact

. Alter

air can be bubbled thr

ough the

eactor

. If

ou look at the f

you will see that the bioreactor has an agitator system, an oxygen

system and a foam control system, a temperature control syst

control system and sampling ports so that small volumes of the

can be withdrawn periodically.

timal conditions for achieving the desired

roduct b

optimum growth conditions (temperature, pH, substrate, salts, vi

oxygen

The most commonly used bioreactors are of stirring type, w

shown in F

ure 11.7.

Figure 11.7

(a) Simple stirred-tan

bibi

ctor; (b) Sparged stirred-tank bioreactor through

sterile air bubb

are

arged

(

)

(

)