BTNY-MM: XII 11.

BIOTECHNOLOGY: PRINCIPLES & PROCESSES

Biotechnology is the technological exploitation of biological  Stanley Cohen & Herbert Boyer (1972) constructed the first
processes for the benefit of mankind. recombinant DNA.
 Biotechnology has two aspects or views- They linked a gene of antibiotic resistance from the plasmid of
Salmonella typhimurium with a plasmid of E.coli and transferred
Modern Biotechnology / into E.coli.
Classical Biotechnology rDNA technology /
Genetic engineering
 It includes the processes  Modifying the organism
based on the natural genetically and then is used
capabilities of microbes. for obtaining the products
e.g.: making curd, e.g.: In vitro fertilisation,
bread, wine Preparation of vaccines
using rDNA technology,
gene therapy
 Merits of genetic engineering over traditional hybridisation
Hybridisation techniques lead to inclusion and multiplication of
undesirable genes along with desired genes.
Genetic engineering helps to isolate and introduce only desirable
genes into the target organism.

ii. Removal of impurities from the DNA

Explanation:-  RNA - removed by treating with ribonuclease
I. Isolation of a desired DNA fragment  Proteins - removed by treating with protease.
i. Breaking the cell wall. Other molecules can be removed by appropriate treatments.
Depending on cell which has to be lysed, different lytic enzymes
are used. iii. Separation of purified DNA
For bacterial cell → lysozyme Add chilled ethanol to precipitates out the purified DNA (can be
For plant cells → cellulase seen as collection of fine threads in the suspension). This can be
For fungal cell → chitinase separated by spooling.

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iv. Cutting of DNA at specific location using Restriction  Polymerase Chain Reaction is technique used to amplify
Endonuclease. (synthesis multiple copies) the gene of interest
Cut the purified source DNA as well as the vector DNA with a Kary Mullis (1983) developed this technique.
specific restriction enzyme. Requirements-
Restriction Enzymes  DNA template whose copies are to be produced
 Free nucleotides
These enzymes actually occur in bacteria to ‘restrict’ the  2 sets of primers - Primers are small chemically synthesized
replication of attacking bacteriophages by identifying their oligonucleotides that are complementary to 3’ end of template.
introduced phage DNA and cut it into pieces (cleave (Primers provide a free 3’-OH required by DNA polymerase for
phosphodiester bond). the formation of the phosphodiester bond)
 DNA polymerase- Thermostable Taq polymerase (isolated from
 Restriction enzymes belong to a class of enzymes called
a bacterium, Thermus aquaticus) is used.
nucleases. They include exonucleases & endonucleases.
Restriction enzyme Steps: -
Denaturation: The DNA fragment is heated about 940C results
Exonucleases Endonuclease in separation of DNA strands.
Remove nucleotides Cuts at specific Annealing of primer: The solution is allowed to cool to 600C.
from the ends of DNA positions within the DNA During this time, one primer anneal (join) with 3’ end of one
DNA strand and the other primer binds with the 3’ end of its
Action of Endonuclease: complementary strand.
Cuts at specific recognition sequences (often 4/6 base pairs long) Primer extension: DNA polymerase extends the primers by
which are palindromic. adding nucleotides complementary to the template.
Cut leaves a single stranded overhanging stretches portions at each Amplification: The process of replication is repeated.
ends (Sticky ends-they form H-bonds with their complementary cut
counterparts. This stickiness of the ends facilitates the action of the
enzyme DNA ligase).
 The first restriction endonuclease–Hind II from Haemophilus
I
influenzae.
Naming of restriction enzymes: - Eg: EcoRI
st
 1 letter (in capital and italics) - Genus of the II
prokaryotic cell from which enzyme were isolated. E- Escherichia
 2nd & 3rd letters - Indicate species. co- coli
th
 4 letter- The strain name of bacteria. R- RY 13 III
 Following Roman numerals indicates the
order of isolation from the strain. I- first
IV
v. Desired DNA fragment is separated using Gel
electrophoresis.
Because the endonuclease’s recognition sequence is likely to occur
many times within the source DNA, cleavage will produce a large
number of different fragments.
 Of these, desired DNA fragment can be separated by a technique
known as gel electrophoresis. Developed by A.Tiselius.
It separate DNA fragments based on their size, under the influence of - The amplified fragment can be used to ligate with a vector for
electric field. further cloning.
Steps:
a. DNA are forced to move under an electric field through the II. Ligation of the DNA fragment into a vector
agarose gel (medium).Negatively charged DNA will move (preparing rDNA)
towards anode a/c to their size through sieving effect provided by Cloning Vectors:-
the medium. (Hence, the smaller sized fragment move farther). They are the DNA molecules that can carry a foreign DNA segment
b. To see DNA on gel block, it is stained with Ethidium Bromide and replicate inside a host cells.
and when viewed in UV light, it is seen as bright orange coloured  The desired DNA fragments are incorporated into the vector, which
bands. have been cleaved with the same restriction endonuclease as the
 Elusion: The process of extracting separated DNA bands from source DNA, using DNA ligase.
the agarose gel. Desired DNA + Vector = rDNA
Desirable features of a cloning vector-
(a) Presence of Ori (Origin of Replication)
 This is a sequence from where replication starts. A piece of DNA
linked to ori can only replicate within the host cells.
(b) High copy no.
 Copy number is the number of plasmids needed for a host cell.
 When the piece of DNA linked with bacteriophage or plasmid
vectors, it multiplies to the numbers equal to the copy number of
the vector.
(c) Presence of selectable markers.
vi. Amplification of gene of interest using PCR.  Selectable markers are genes which help in differentiating
A single piece of desired DNA fragment obtained by the above transformants and non-transformants. Upon the introduction of
procedures may loss or damaged during the further procedure.
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 foreign gene in this marker sequence, phenotype is loaded & that  Ti plasmid of Agrobacterium tumifaciens
helps in screening whether the cell has been transformed or not. - Tumor inducing plasmid is modified into a cloning vector which
e.g.: Antibiotic resistance gene (such as ampR, tetR), does not produce tumour, but is able to use the mechanisms to
Enzyme producing gene (such as lac Z gene which produces deliver genes of interest into plants.
-galactosidase)
(d) Presence of unique recognition sequence in cloning sites.
 It should have at least one recognition site for restriction enzymes
in order to link the desired DNA,.
(Presence of more than one recognition sites generates several
fragments, which complicates the gene cloning).

Types of Vectors:
1. Plasmids
These are circular extra-chromosomal DNA of bacteria.
e.g.: pBR322, Ti plasmid
 pBR322
- Plasmid of Boliver and Rodriguez. Numeral ‘322’ is assigned to
distinguish the plasmid from others that are also studied in their
laboratory. 2. Retroviruses
Retroviruses in animals have the ability to transform normal cells
into cancerous cells. Disarmed (incapable of causing disease)
retroviruses are used to transfer the rDNA into the animal cells.
3. Bacteriophages

III. Transferring the rDNA into the host

(transformation)
Gene transfer may be done by 2 ways-
A. Vector through gene transfer
pBR322 consists of- B. Vector less gene transfer
- 8 restriction enzyme recognition sites
- ori VECTOR LESS Gene Transfer-
- rop (Replication of Plasmid Protein) is a gene synthesising the The process of transfer of genes directly into the host cells. It can
proteins necessary for replication of plasmids. be done in 3 methods-
- 2 selectable markers – ampicillin and tetracycline resistance
gene.
Restriction endonuclease on pBR322, their source and
restriction sites
Prokaryote from which Recognition
Enzyme
enzyme is extracted sequence

Haemophilus influenzae 5’- AAGCTT -3’

Hind III
D-strain 3’- TTCGAA- 5’

Escherichia coli RY13 - 5’- GAATTC -3’

EcoR I
strain 3’- CTTAAG- 5’ (1st-way) Heat-shock method
Since DNA is a hydrophilic (water soluble) molecule, rDNA
cannot pass through cell membranes (lipid soluble). In this, making
Bacillus amyloliquefaciens 5’- GGATCC -3’
BamH I the host cell competent (ready to take up rDNA)
H-strain 3’- CCTAGG- 5’
Steps:-
a. Treating bacterial cell with a specific concentration of a divalent
5’- CGATCG -3’ cation (such as Ca2+)
Pvu I Proteus vulgaris b. Incubate the cells with rDNA on ice.
3’- GCTAGC- 5’
c. Placing them briefly at 420C (heat shock)
This makes temporary holes in the cell surface enables the bacteria
Pvu II 5’- CAGCTG -3’ permeable to recombinant plasmid to enter.
” 3’- GTCGAC- 5’ d. Put them back on ice.
(2nd-way) By micro-injection
5’- GTCGAC -3’  Micro needle of 1-2µ is used to inject DNA directly into the
Sal I Streptomyces albus
3’- CAGCTG- 5’ nucleus of an animal cell.
(3rd-way) By gene gun method
5’- CTGCAG -3’  In this, DNA coated micro-particles of gold or tungsten are
Pst I Providencia stuartii shoted with high velocity (1400ft/s) to plant cells.
3’- GACGTC- 5’

5’- ATCGAT -3’

Cla I Caryophanon latum
3’- TAGCTA- 5’

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 Selection of transformants
Selection of transformed cells from non-transformed ones with the
help of selectable markers & its insertional inactivation.
Selection is of 2 types:-
A. Using antibiotic resistance gene as marker
o The ligation of foreign DNA is carried out at a restriction site
present in 1 of the 2 antibiotic resistance genes.
E.g. Normal E.coli cells do not carry resistance against antibiotics
(non-transformant).
If pBR322 is added to E.coli then they are transformant and
they can grow on both ampicillin and tetracyclin medium. Thus
these marker genes help in differentiating transformants from the
non-transformants
If a foreign DNA is added at the BamH I site of tetR gene, this
gene get inactivated due to insertion of foreign DNA (this is
called insertional inactivation). Hence the bacteria/
transformant will not grow on tetracycline, but will grow on IV. Culturing the host cells
ampicillin.  To produce large quantities of products, the bioreactors are used
where large volumes (100-1000 litres) of culture can be processed.
 A bioreactor provides the optimal growth conditions (temperature,
pH, substrate, salts, vitamins, oxygen) for achieving the desired
product.

Simple Stirred-tank reactor

Cylindrical reactor having-
 Stirrer / An agitator system- Facilitates even mixing of the
reactor contents
 An oxygen delivery system
 A foam control system
 A temperature control system
 pH control system
 Sampling ports (for periodic withdrawal of the culture).

B. Using enzyme producing gene as marker

On the basis of colour production in presence of chromogenic
substrate, the transformant and non- transformant can be
differentiated.
o This method involves insertional inactivation of lac-Z gene and Sparged stirred-tank reactor
also known as Blue-white selection method.  It is a stirred type reactor in which air is bubbled at the bottom
Normal bacteria (non- transformant) contain Lac-Z gene on of the tank through a porous ring called spranger.
their plasmid and Lac-Z gene is responsible for the production of
enzyme β –galactosidase.
β –galactosidase
White substance Blue colour
If the plasmid in bacteria have no an insert it gives blue coloured
colonies
Tranformant bacteria in which foreign DNA is inserted within
the Lac-Z gene, it gets inactivated and thereby no enzyme β –
galactosidase is produced. Such colonies remain white. These are
identified as recombinant colonies.
V. Extraction of the desired product
The product obtained from bioreactor is not in pure form.
 The extraction & purification of desired product from the
culture medium is known as Downstream processing.
Procedure-
Step 1. Separation and purification of gene products.
Step 2. Addition of suitable preservatives.
Step 3. Goes thorough clinical trials (in case of drugs).
Step 4. Quality control test.

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