Biotechnology:

Principles and Processes

POINTSTO
REMEMBER
REMEMBER
POINTSTO
1, Biotechnology
Botechnology deals with microorganisms, plant or animal cells or their enzymes to produce products
humans.
and processes useful to
. The term "Biotechnology" was given by Karl Ereky (1919).
According to European Federation of Biotechnology (EFB), biotechnology is the integration of natural
science and organisms, cells, parts thereof,and molecular analogues for products and services.

2.Principles of Biotechnology.
I The two core techniques that developed modern biotechnology are:
DNA/RNA and their
() Genetic engineering which is nodification of chemical nature of
introduction intoanother host organism,to change the phenotypic characters of the host.
desired microbes
(i) Sterilisation methods to maintain growth and manipulation of only the
biotechnological products like antibiotics,
or cells in large quantities, for the manufacture of
vaccines, enzymes, etc.
The basic steps in genetic engineering include:
() identification of DNAwith desirable genes.
recombinant DNA (rDNA).
(ü) introduction of the DNA into host to form
(i) maintenance of DNAin host and gene cloning.
(iv) gene transfer.
Boyer constructed the first recombinant DNA.
n 19/2,Stanley Cohen and Herbert fashion and
of E. coliwhich cut DNA in particular
erbe& Boyer worked on restriction enzymes restricted ends were ligated with desired pieces of
Produce sticky ends on both the strands. These
DNA,. developed
plasmid DNA loating freely in cytoplasm of bacterial cells. He also
aey Cohen studied
and reinserting them in other cells.
*etnod of removing plasmids from the cell bacteria and then linked the gene with plasmid
They isolated antibiotic resistant gene from plasmid of
where it could replicate using the new host's LDNA polymerase enzyme
COrporated into Ecoli,
and make multiple copies.
recombinant DNA:
Steps carried out in constructing first antibiotic resistance gene by cutting out a piece
of DNA from
() The first step is to isolate the
conferring antibiotic resistance.
PaSid which was responsible for
Biotechnology: Principles and Processes 407
REMEMBER
TO
POINTS
(ii) lsolation of genes takes place with the help of molecular sciossors restriction enzymes.
(ii) The Cut piece of DNA wasthen linked with a native plasmid of Salmonella typhimorium that
Plasmid DNA acts as vector totransfer the piece of DNA attached to it then trarnsferred

E. Coli for gene multiplication.

3.Tools of Recombinant DNA Technology

DNA technology are:
The key tools required for the recombinant(ii) Polymerase enzymes
(1) Restriction enzymes
(iv) Vectors
(ii) Ligases
(o) Host organism/cel.
4. Restriction Enzymes
restriction enzymes are called "molecular scissors" and are responsible for cutting DNA
The
They are present in bacteria to provide a type of defence mechanism called the "restriction-

modification system".endonuclease, HindIL, was isolated by Smith, Wilcox and Kelley (1968) from
The first restriction
Haemophilus influenzae bacterium. It was used to cut DNA molecules at a particular point
recognition sequence.
recognising a specific sequence of six base pairs, known as the
Naming of Restriction Enzymes
The first letter is derived from the genus name and the next two letters from the species name of sL.
prokaryotic cell from where the enzymes are extracted. The roman numbers, following the nams
show the order in which the enzymes were isolated from the bacterial strain.
For example, EcoRI is derived from Escherichia coli RY13, HindI from Haemophilus influenzae RA,
BamI from Bacillus amyloliquefaciens H, EcoRII from E. coli R245, etc.
Restriction enzymes belong to aclass of enzymes caled nucleases and are of two types:
() Exonucleases--cut DNA at the ends.
(ü) Endonucleases--cut at specific positions within the DNA.
The recognition sequences of endonucleases are palindromic, i.e., the sequence of base pairs read the
same on both the DNA strands, when orientation of reading is kept same, eg.,
GAATTC.
3 CTTAAG. 5

Mechanism of Action of Endonucleases

Every endonuclease inspects the entire DNA sequence for the palindromic recognition sequence.
On finding the palindrome, the endonuclease binds to the DNA.
It cuts the opposite strands of DNA in the sugar-phosphate backbone; alitle away from the centre of
the palindrome sites but between the same bases on both strands.
This results in the formation of single stranded overhanging stretches at the end of each strand cauu
sticky ends.
The sticky ends facilitate the action of the enzyme DNA ligase by readily forming hydrogen bonds
with complementary strands.
In genetic engineering, DNA from different:i sources are cut withthe same restriction enzymes sothat
both DNA fragments have same kind of sticky ends.
These sticky ends are complementary to each other and thus can be joined by DNA ligase(end-to-
end).
408 Xam idea Biology-XIl
 The enzyme cuts both DNA
EcoRl
Gand cuts the DNA
strands at the same site
A only when the between bases
GAATTCDNAis present
Foreign sequence
In the DNA
Vector DNA
REMEMBER
POINTSTO
EcoRI

Sticky end

Sticky end
DNA fragments join at sticky
ends

Recominant DNA
Fla. 9.1 Steps in formation of
recombinant DNA by action of restriction
endonuclease enzyme EcoRI
and Isolation of DNA
S. Separation Fragments (Gel Electrophoresis).
Celelectrophoresis is a technique for separating DNA fragments based on their size.
Firstly, the sample DNA is cut into fragments by restriction endonucleases.
Wells
DNA
bands
Largest Smallest

Cathode (-ve)+
+Anode (+ve)

Fig. 9.2 Atypical agarose gel electrophoresis showing migration of undigested (lane 1) and
digested set of DNA fragments (lane 2to 4)
NTagments being negatively charged can be separated by forcing them to move towards the
anode under an electric field through a medium/matrix.
Oyused matrix is agarose, which is a natural linear polymer of D-galactose and 3, 6-anhydro
L-galactose which is extracted from sea weeds.
The DNA fragments separate-out (resolve) according to their size because of the sieving property of
agarose gel." Hence,smaller the fragment size, the farther it will move.
The Separated DNA tragments are visualised after staining the DNA with ethidium bromide

fol owed by exposure to UV radiation.

I The
DNA !fragments are seern
The Separated bands of DNA as Orange
are cut
coloured bands.
out and extracted from the gel piece. This step is called elution.
The puurified DNA ITagments are used to form recombinant DNA which can be joined with cloning

vectors.
Biotechnology: Principles and Processes 409
POINTS
TO
REMEMBER
into
6. Cloning Vectors foreign DNAsegment the host cell.
that cancarry a
DNA molecules
The vectors arethe
circular extra-chromosomal DNA.
Vectors may be:
autornomously replicating bacteria. Bacteriophages
(a) Plasmids: These are viruses that infectwithin bacterial cells.
because of
Copy Bacteriophages: These
(6) number: It is defined
are
as high copy number
the number of copies of vectors present in a cell. It varies from
their high
have very
humber per cell

1-100 copies per cell.

vector.
vector is the plasmid 1977 by Boliver and
The best known
pBR322 is the first
vector developed in
artificial cloning Rodriguez from
E. coli plasmid. facilitate cloning into a vector:
features are required to EcoR I
The following Cla Hind |l
() Origin of replication (ori)
DNA sequence that is responsible for Pvul:
This is a of DNA when linked
initiating replication. Any piece cells. Pst I
replicate within the host ampR BamH
to this sequence can linked
copy numbers of the tefR
ori also controls the
target DNA, it should be
DNA. For many copies of pBR322
copy
cloned inavector whose origin supports high Sal|
number.
ori rop
(ii) Selectable marker
which contain the
" It helps to select the host cells
vector (transformants) and eliminate the non Pvu l
transfornmants.
Transformation is defined as the procedure by which Fig. 9.3. E. coli cloning vector pBR322
showing restriction sites (Hindll, EcoRl.
apiece of DNAis introduced into a bacterial cell. BamHl,Sall, Pvull, Pstl, Clal), ori and
Genes encoding resistance to antibiotics like antibiotic resistance genes (amp" and tet
rop codes for the proteins involved in the
ampicillin, chloramphenicol, tetracycline or
replication of the plasmid.
kanamycin, are useful selectable markers for E. coli.
The normal E. coli cells do not carry resistance against any of these antibiotics.
(ii)Cloning sites
To link the alien DNA, the vectors require very few (mostly single) recognition sites ror u
restriction enzymes.
More than one recognition sites within the vector, can complicate the gene cloning asitwill
generate several fragments.
Ligation of alien DNA can be carried out at a restriction site present in one of the two antibiotic
resistance genes.
(iv) Vectors for cloning genes in plants and
" There are several vectors which
animals
are used for cloning genes in plants
and
" In plants, the tumour inducing plasmid (Ti) of Agrobacterium tunefaciens is animals.
used as acloningvector.
" A. tumefaciens is a pathogen of
several dicot plants.
It delivers a piece of DNA known as "T-DNA' in the Ti plasmid which transforms normalplant
cells into tumor cells to
produce chemicals against pathogens.
410 Xam idea Biology-XIl
 Retrovirus,adenovirus, papillomavirus are
also now used as cloning
theirabilityto transform
normal cells into vectors in animals because
usedto transfer desirable genes to animal cells.cancerous cells. They are first
of
disarmed and then
Selectionoffrecombinants formed can be done by one of the
following
REMEMBER
POINTS TO
Inactivationof antibiotics
methods:
Ifaforeigm DNA ligates at the BamHI site of tetracycline resistance gerne in the vector pBR322, the
recombinant plasmid loses the tetracycline resistance due to
insertion of foreign DNA.
Itcanstill be selected out from non-recombinant ones by plating the trarnsformants on ampicillin
containingmedium.
Thetransformantss growing on ampicillin containing medium are then transferred on to a medium
containing tetracycline.
The recombinants can grow in ampicillin containing medium but not on that containing
tetracycline whereas non-recombinants can grow on the medium containing both the
selected. antibiotics
and thus recombinants are
(b)Alternative Selectable Marker (Insertional inactivation)
e On the basis of colour production in the presence of chromogenic substrate, the recombinants
and non-recombinants can also be differentiated.
Here a recombinant DNA is inserted within the coding sequence of an enzyme B-galactosidase,
which results into inactivation of the enzyme and hence there is no conversion of substrate to
products.
Hence, the bacterial colonies having transformed plasmid, shows no colouration while those
without inserted plasmid form blue colour colonies.

2.Competent Host (For Transformation with Recombinant DNA).

DNA being ahydrophilic molecule, cannot pass through cell membranes.
Therefore, the bacteria should be made competent to accept the DNA molecules.
I Competency is the ability of a cell to take up foreign DNA.
The cellis made competent by the following methods:
() Chemical method (1) Physical method
(i) Chemical method
" The cell is treated with specific concentration of a divalent cation such as calcium to increase
pore
size in cell wall.
them briefly at 42°C
"The cells are incubated with recombinant DNA on ice, followed by placing
and then putting it back on ice. This is called heat shock treatment.
" The bacteria now take up the recombinant DNA.
(i) Physical methods
The physical methods include
into the nucleus of an animal cell
MICro-injection method: Recombinant DNAis directly injected
by micro-pipettes. or
bombarded with high velocity micro-particles of gold
oroistiCor gene gun method: Cells are
tungsten coated with DNA in plants.
rDNA like Agrobacteriumtumefaciens.
Disarmed pathogen vectors are also used to transfer
411
Biotechnology: Principles and Processes
POINTS
TO
REMEMBER
Recombinant DNA Technology
8. Process of
involves the following steps:
Kecombinant DNA technology
(i) Isolation of DNA. endonucleases.
DNAby restriction
() Fragmentation of
fragment.
(ii) Isolation of a desired DNA
interest.
(r0) Amplification of the gene of
fragment into a vector.
(v) Ligation of the DNA the host.
recombinant DNA into
(vi) Insertion of scale.
on a suitable medium at a large
(i)Culturing the host cells
(vii) Extraction of the desired gene product. finished product, ready for marketing.
of the products as
(1*) Downstream processing
Vector
Foreign DNA DNA
DKXDDDKDDD (Plasmid)
both foreign
Same restriction enzyme cuting point
DNA and vector DNA at specific

DI DDDD
D
Ligases join forelgn
DNA to plasmid

Transformation

E. coli
Cells divide

Fig. 9.4 Diagrammatic representation of recombinant DNA technology

() Isolation of the genetic material (DNA)

macromolecules.
To cut DNA with restriction enzymes DNA should be purified, free from other
The bacterial/plant/animal cellis broken down by enzymes to release DNA, along with
proteins, polysaccharides and lipids.
Bacterial cell is treated with enzyme lys0zyme.
Plant cell is treated with enzyme cellulase.

412 Xam idea Biology-Xli

 Fungalcellis treated with chitinase.
RNAisremoved by treatment with
eprotease. ribonuclease and proteins are removed by
treatment with
Afterseveral treatments, the
purified DNA is precipitated by adding chilled
ethanol.
Cutingof DNA at specific locations
i DNAis cut using restriction
e The
enzymes.
purified DNAis incubated, with the specific
The
to act.
restriction enzyme at conditions optimum for
theenzyrme
Processis repeated with vector DNA also.
e
Isolationoffdesired DNA fragment
)
Using agarose gell electrophoresis, the activity of the restriction enzymes can be checked.
Sincethe DNA is negatively charged, it moves towards the positive electrode or anode and in the
process, DNA fragments separate out based on their sizes.
. The desired DNA fragment iseluted out.

)Amplification of gene of interest using PCR

The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA
sequences is carried out in vitro.
This technique was developed by Kary Mullis in 1985, and for this, he received Nobel Prize for
Chemistry in 1993.

wW 5'
Denaturation
3'

Annealing
5'

DNA polymeraso
5
3 3
5
Elongatlon 5'
3 m 3

reaction (PCR)
Flg. 9.5, Polymerasechain

413
Biotechnology: Principles and Processes
REMEMBER
TO
POINTS
be amplified.
" Requirements for PCR:
double-stranded
(b) Primers: Small chemically LDNA
sVnthesised thatneeds toof
oligonucleotides about 10 18 nucleotides that are
The
DNA template:
() Enzyme: Two commonly of template DNA.
Complementary to a region used enzymes are Taq polymerase (isolated from thermophikc

Vent polynerase (isolatedfrom Thermococcus litoralis).

bacterium, Thermus aquaticus) and
" PCR is carried out in the following three steps:
(a) Denaturation
The double-stranded DNA is denatured bysubjecting it to high temperature of(94°C for 15
seconds. Each separated single stranded strand now acts as templatefor DNA synthesis.

(b) Annealing the 3' end of each separated strand.

added which anneal to
Two sets of primers are
Primers act as initiators of replication.
(c) Extension
DNA polymeraseextends the primers by adding nucleotides complementary tothe template
provided in the reaction.
used in the reaction which can il
Athermostable DNA polymerase (Taq polymerase) is
the high temperature of the reaction.
copies of desired DNA
All these steps are repeated many times to obtain several
(v) Ligation of DNA fragment into a vector
The vector DNA and source DNA are cut with the same endonuclease to obtain sticky ends.
" These are then ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form
arecombinant DNA.

(vi) Insertion of recombinant DNA into the host cell/organism

" Introduction of ligated DNA into recipient cells occurs by several methods, before which the
recipient cells are made competent toreceive the DNA.
Ifrecombinant DNA carrying antibiotic resistance (e.g., ampicillin) is transferred into E. coli
the host cell is transformed into ampicillin-resistant cells. cells,
" The ampicillin resistant gene in this case is called a
selectable marker.
" On growing transformed cells on agar plates
and others will die. containing ampicillin, only transformants will grow
(vii) Culturing the host cells
The transformed host cells are grown in
The DNA gets multiplied and appropriate nutrient medium at optimal conditioS
expresses itself to form desired product.
(viii) Extraction of desired gene
When a protein encoding product
gene
is
protein. expressed in aheterologous host, it is called a recombinant
The cells having genes of
" On small scale, the cells
interest can be grown on a small
are grown on cultures in scale or on a large scale.
extracted purified by different
and laboratory and then the
expresseu
proteinis
e On large
scale, cells are groWnseparation techniques.
added from onethe in a
side, to maintain cells
collected from the other side. in continuous culture system in which fresh mediumis
In Jarge scale exponential growth phase and the desired protein
method, larger biomass is
produced which leads to high yield.
414 Xam idea Biology-XI
 NDownstreamprocessing
processesto which a product is
Allthe
arecalled downstream processing.
Itincludes:
REMEMBER
POINTSTO
subjected to before being marketed as afinished
prodt
(a) Separation ofthe product from the reactor.
Purification of the product.
() Formulation of the product with suitable preservatives.
(d) Quality control testing and clinical trials in case of drugs.
9,Bioreactors
Bioreactors are vessels of large volumes (100-1000 litres) in which raw
products. materials are biologically
converted into specific
lt providesallthe optimal conditions for achieving the desired product by providing optimal growth
Conditions like temperature, pH, substrates, salts, vitamins and
oxygen.
, Stirred-tank bioreactors are commonly used bioreactors.

Increased
surface
Acid/Base area for
Motor Gas
for pH Oxygen
control transfer entrainment
-Foam
braker
Flat bladed
Steam for impeller
sterilisation
-Culture
broth
Bubbles
dramatically
increase the
-Sterile air OXygen
(a) transfer area (b)

Fg. 9.6. (a) Simple stirred-tank boreactor; (6) Sparged stirred-tank bioreactor through which sterile air
bubbles are sparged.

ese are cylindrical with curved base to facilitate (i) proper mixing of the contents, (2) maintain
Oxygen availability throughout the bioreactor.
tank reactor has (i) better temperature and pH control, (i) foam control system to prevent
foam and shearing damage to cells due to agitation, (ii) system sterilisation, and (iv) provision to
Withdraw small volumes of cultures periodically.
Abioreactor has the
) An following componentS:
agitator
") An oxygen system
(i) Foam delivery system
control
0) Temperature system
(u) pH control system
(ui) control ports
systemto withdraw cultures periodically
Sampling
The stirrer mixes
the available throughout the bioreactor.
Sparged stirred-tankcontents
reactor iand makes oxygen
s astirred type reactor in which air is bubbled.
Biotechnology: Principles and Proce