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Oct 23, 2013 (3 years and 9 months ago)

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PROJECT DESIGNERS


G R KRISHNA CHAND AVATAR ROLL
-

8
GINNGAIHLIAN ROLL
-

7

CERTIFICATE


T
his is to certify that this project entitled “BIOTECHNOLOGY DEFINITION,
PRINCIPLES AND PROCESSES” has been designed by both of us :


Ginngaihlian R. No
-


G R Krishna
Chand

Avatar R. No.
-



to the best of our abilities for the partial fulfillment of the requirements of AISSC
Examination 2012
-
13.


It is a comprehensive work carried out under the supervision of Ms Lois Varghese
and her satisfaction.




DATED : J C LALSANGZUALA


PLACE : PRINCIPAL


JNV,
Thenzawl




CERTIFICATE


T
his is to certify that this project entitled “
BIOTECHNOLOGY
DEFINITION, PRINCIPLES AND PROCESSES
” assigned to


Ginngaihlian R. No
-


G R Krishna
Chand

Avatar R. No.
-



of Class XII (Science) for the partial fulfillment of the requirements of AISSC
Examination 2012
-
13.


It is a comprehensive work carried out under my supervision and to the
best of my satisfaction.




DATED :
Mrs

Lois Varghese


PLACE : PGT (Biology)


JNV,
Thenzawl



ACKNOWLEDGEMENT


At the first instant, we would acknowledge
Lord Almighty

for His
incessant eternal support and encouragement. We always were brimming
with enthusiasm with His divine blessings throughout the process of the
completion of the project.


We are deeply indebted to our guide and mentor Ms. Lois Varghese,
PGT (Bio) for the constant help rendered by her. Her ever readiness to
She was always with us in heart and soul during the progression of this
work.


Not underestimating, we are thankful to our peers for the support
they lent to us.


Last but not the least, we pay our fathomless gratitude to our parents
for the mental zeal they bestowed on us. They whole
-
heartedly provided
all the possible help to elevate this project to the highest level.


PLACE :
Ginngaihlian


DATED: Roll No:

PLACE: G R K C Avatar

DATED: Roll No:

PRINCIPLES AND
PROCESSES OF

BIOTECHNOLOGY

DEFINITION


Biotechnology deals with techniques of using live organisms or their
components or enzymes from organisms to produce products and
processes useful to humans.



The European Federation of Biotechnology (EFB) furnishes its implication
as:


The integration of natural science and organisms, cells, parts thereof, and
molecular analogues for products and services.

PRINCIPLES OF MODERN BIOTECHNOLOGY

1. GENETIC
ENGINEERING

The technique of altering chemistry
of genetic material, i.e., the nature of
DNA/RNA and/or introduction of it
into host organisms and thus
changing the phenotype of host
organism
.

2. CULTURE
TECHNIQUES

It facilitates the growth and
multiplication of only the desired
microbes or cells in large number,
under controlled conditions, in
large quantities for the
manufacture of biotechnology
products like antibiotics, vaccines
or enzymes.

TECHNIQUES OF GENETIC ENGINEERING

The technique of genetic engineering includes :

(i)
Creation of recombinant DNA

(ii)
Use of gene cloning, and

(iii)

Gene transfer. With this technique, we can
isolate and introduce only the desired genes
without introducing undesirable genes into
target organisms.

WHAT IS CLONING?


Cloning or making multiple copies of any
template DNA needs that an alien DNA is
linked with the origin of replication (a desired
DNA sequence responsible for initiating
replication) so that alien (foreign/desired)
pieces of DNA can replicate and multiply itself
in the host organism.

CREATION OF RECOMBINANT DNA


The first recombinant DNA was constructed by
Stanley Cohen
and
Herbert Boyer
in 1972 by;

(i)
Isolating the antibiotic resistance gene by cutting out a piece of
DNAfrom

a plasmid which was responsible for providing antibiotic
resistance. The cutting of DNA at specific location was done by
molecular scissors


restriction enzymes
.

(ii)
The cut pieces of DNA were then linked with the plasmid DNA.

(iii)
The plasmid DNA acts as vector to transfer the pieces of DNA
attached to it, to the recipient host bacterium.

(iv)
Linking of antibiotic resistant gene (alien DNA) with plasmid DNA
(vector) was possible with enzyme
DNA
ligase
,
which joins the cut
ends of DNA molecules.

(v)
Hence, a new circular, autonomously replicating DNA created
in vitro
is
formed called as
recombinant DNA.

(vi)
It replicates using the new host’s DNA polymerase enzyme and makes
multiple copies.
The ability to multiply copies of antibiotic resistant
gene in E. coli is called
cloning.


TOOLS OF RECOMBINANT DNA TECHNOLOGY

(i)
Restriction enzymes

(ii)
Polymerase enzymes

(iii)
Ligases

(iv)
Vectors

(v)
Host organisms

RESTRICTION ENZYMES


Restriction enzymes belong to a class of enzymes
called nucleases. They are of two types:




(a)
Exonucleases

:

Remove nucleotides from the
ends/terminals of DNA.



(b)
Endonucleases :
Cut the DNA at specific
position of N
-
bases anywhere in the length except
ends.

RESTRICTION ENDONUCLEASES
-

AN INTRODUCTION


Linn
and
Arber
(1963) isolated two enzymes from
E. coli
responsible for restricting the growth of bacteriophage, one
of them added methyl group (CH
3

-
) to DNA while the other
cut the DNA into segments and is called
restriction
endonucleases.


Smith,
Willox

and
Kelley
(1968) isolated and
characterized the first restriction endonuclease from
Haemophilus influenzae
bacterium called it

Hind
-
II. Hind
-
II

always cut DNA at a particular site/point by recognizing
a specific sequence of six base pairs called
recognition
sequence
for
Hind
-
II.
Today more than 900 restriction
enzymes have been isolated from over 200 strains of
bacteria, each of which recognizes different recognition
sequences.

RESTRICTION ENDONUCLEASES (
CONTD
)


The recognition sequence is
palindromic

where the sequence of base pairs
reads the same on both the DNA strands when the orientation of reading is
kept the same, i.e., 5’→3’ or 3’→5’ direction e.g. 5’
-

GAATTC
-

3’ 3’
-

CTTAAG
-
5’.


Each RE function by ‘inspecting’ the length of a DNA and then binds to the
DNA at recognition sequences.


RE cut the two strands of DNA double helix at specific points in their sugar
-
phosphate backbone, a little away from the centre of
palindromic

site, but
between the same two bases on both the strands. As a result single stranded
portions are left at the end of DNA in each strand called
sticky ends.


Unwanted self ligation of vector DNA molecules by removing phosphate
group from the 5’ end of a DNA molecule, leaving a free 5’ hydroxyl group,
using alkaline phosphate from bacteria (BAP) or calf intestine (CAP).


These single stranded DNAs in each strand form hydrogen bonds with their
complementary counterpart (single strands of alien DNA). This stickiness of
the ends facilitates the action of enzyme DNA
ligase
. Hence RE are used in
genetic engineering to form “Recombinant molecules of DNA”

RESTRICTION ENDONUCLEASES (
contd
)


When cut by the same RE, the resultant DNA fragments have the same
kind of sticky ends and these can be joined together using DNA
ligase
.



NOMENCLATURE OF RESTRICTION
ENZYMES


The first letter of the name comes from genus and the next two letters from the
name of the species of the species of the bacterium (prokaryotic cell) from
which they are isolated.



The next letter comes from the strain of the bacterium.



The Roman number following these four letters indicate the order in which
enzymes were isolated from that strain of the bacterium, e.g.


(a) Eco R
-
I is isolated from
Escherichia coli
RY 13


(b) Hind
-

II is from
Haemophilus influenzae


(c) Bam H
-
I is from
Bacillus amyloliquefaciens


(d) Sal
-
II is from
Streptomyces albus.

CLONING VECTOR


Cloning Vector includes plasmid and
bacteriophages
, which have the ability
to replicate within bacterial cells and independent of the control of
chromosomal DNA. A vector should have very high copy number of their
genome within their bacterial cell so that a linked alien piece of DNA, can
multiply its number equal to the copy number by vector employed.



Vectors used at present are engineered in such a way that they help easy
linking of foreign/alien DNA and selection of recombinant from non
-
recombinant.



CLONING VECTOR

FEATURES OF VECTOR


Following are the features that are required to facilitate cloning in a vector.


1. Origin of replication


2. Selectable marker


3. Cloning site (
Recognisation

vector)


4. Small size of vector


Origin of replication (
ori
): This is the sequence from where replication starts and
a piece of DNA linked to this sequence can be made to replicate within host cells.
This sequence also controls the copy number of vector DNA or linked alien DNA.


Selectable marker
: It helps in
identifying and
eliminating non
-
transformants

and
help in selecting those host cells which contain the vector, i.e.,
transformants
.


Cloning sites
: The vector should have a few or
atleast

one unique recognition site,
to link the foreign/alien DNA( to be use by RE). presence of one recognition site is
preferable . the ligation of a alien DNA is carried out at restriction site present in
one of the two antibiotic resistant genes.





In plants, the Ti
-
plasmid (Tumour inducing plasmid)
of bacterium
Agrobacterium

tumefaciens


has been
modified (does not cause tumour ) is used as a
cloning vector .



Similarly, retroviruses also make the normal animal
cells into cancerous cells. The retroviruses have also
been disarmed and are now used to deliver desirable
genes into animal cells.


Once a gene or a DNA fragment has been
ligated

into
a vector, it is transferred into a bacterial, plant or
animal host.

PROCESSES OF RECOMBINANT DNA
TECHNOLOGY



IT INVOLVES THE FOLLOWING STEPS :


(i)
Isolation of DNA

(ii)
Fragmentation of DNA by REs

(iii)
Isolation of desired DNA fragment

(iv)
Amplification of the gene of interest

(v)
Ligation of DNA fragment into vector

(vi)
Transferring the recombinant DNA into host

(vii)
Culturing the host cells in a culture medium at large scale

(viii)
Extraction of desired products.

PROCESSES OF
RECOMBINANT DNA
TECHNOLOGY


IN DETAIL






1.
ISOLATION OF DNA




DNA is enclosed by membranes along with other macromolecules such as
RNA, proteins polysaccharides and lipids.


(a) The membrane is dissolved by treating the bacterial cells/plant or animal
tissue with enzymes such as
lysozyme

(bacteria),
cellulase

(plant cell),
chitinase

(fungus).


(b) Long DNA molecules are
interwined

with
histone

proteins. RNA can be
removed by treating with
ribonuclease

whereas the proteins can be removed
by proteases.


(c) Other molecules can be removed by treating by appropriate treatments
and now purified DNA precipitates out after the addition of chilled ethanol.


(d) It is seen as collection of fine thread in the suspension.

DNA that separates out can be removed by spooling

2.
CUTTING DNA AT SPECIFIC LOCATION





(a) Fragmentation of DNA is carried out by incubating purified DNA
molecules with restriction enzyme at optimal conditions of temperature and pH
for that specific enzyme.


(b)
Agarose

gel electrophoresis technique is employed to check the
progression of restriction enzyme digestion.


(c) The similar process is repeated with vector DNA.







3. SEPARATION AND
ISOLATION OF DNA
FRAGMENTS



(a) DNA fragments being negatively charged,
can be separated by forcing them to move
towards anode under an electric field through a
medium/matrix (
agarose
). The smaller the
fragment size, the farther it moves.


(b) DNA fragments can be visualized by
staining by staining DNA with
ethidium

bromide followed by exposure to UV
radiations. Bright orange
colour

bands in DNA
became prominent in the gel. The separated
bands of DNA are cut out from gel and
extracted from the gel piece. This step is
known as
elution.


(c)
Purified DNA fragments are used for
reconstructing recombinant DNA by joining
them with cloning vectors.


4. AMPLIFICATION OF GENES OF INTEREST USING
PCR

a)
PCR stands for
polymerase chain reaction.

In this reaction, multiple copies
of gene /DNA of interest are
synthesised

in vitro using two sets of
primers.

b)
The enzyme DNA polymerase.

c)
The enzyme extends the primer using the nucleotides provided in the
reaction and the genomic DNA as template.

d)
Continued DNA replication of segment of DNA can be amplified to
approximately 1 billion times i.e., 1 billion copies are made.

e)
Repeated replication is possible by the use of
thermostable

DNA
polymerase (isolated from bacterium
Thermus

aquaticus

) which remains
active during high temperature induced
denaturation

o double stranded
DNA.

f)
Amplified fragment can be used to
ligate

with vector for further cloning.



5.
JOINING OF DNAs


(a) After cutting DNA with specific RE, the cut out gene of interest from the
source DNA and the cut vector with space are mixed and
ligase

is added.


(b) This results in the preparation of recombinant DNA.


6.
INSERTION OF RECOMBINANT DNA INTO 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.

7. OBTAINING THE FOREIGN GENE PRODUCT



Ultimate aim of all recombinant technologies is to produce a
desirable proteins by expression of recombinant DNA. The foreign
gene gets expressed under suitable conditions, by culturing the host
cell on a suitable medium.

RECOMBINANT PROTEIN


Recombinant

protein

is

produced

if

any

protein

encoding

gene

is

expressed

in

heterologous

host
.




(
i
)

The

transformed

cells

containing

cloned

gene

of

interest

are

grown

in

cultures



BIOREACTORS


Bioreactors are used for processing large volume of culture for
obtaining the product of interest in large quantities. In
bioreactors the raw materials are converted into specific
products, e.g., enzymes.



A bioreactor provides the optimal conditions for achieving the
desired product by providing optimal growth condition such
as:


(
i
) temperature (ii) pH


(iii) substrate (iv) salt


(v) vitamins (vi) oxygen

BIOREACTORS

APPLICATIONS OF

BIOTECHNOLOGY

REASEARCH AREAS IN BIOTECHNOLOGY

Three critical research areas of biotechnology are :




(
i
) Providing the best catalyst in the form of improved organism usually a
microbe or pure enzyme.




(ii) Creating optimal conditions through engineering for a catalyst to act and



(iii) Downstream processing technologies to purify the protein/organic
compound.

BIOTECHNOLOGICAL APPLICATIONS IN
AGRICULTURE

Three options for increasing food production
:


1.
Agrochemical based agriculture

2.
Organic agriculture

3.
Genetically engineered crop based agriculture.



Green Revolution
has succeeded in tripling the yield of crops due to :

(i)
Improved crop varieties

(ii)
Use of better management practices

(iii)
Use of agrochemicals, i.e.
fertilisers
, insecticides and pesticides etc.



GENETICALLY MODIFIED ORGANISMS


(GMO)

GM plants are useful in many ways. Some of their unique features are:


1.
More tolerant to abiotic stresses such as cold, drought, etc.

2.
Have reduced dependence on chemical pesticides (pest
-
resistant
crops).

3.
Reduction in post harvest losses.

4.
Increased efficiency of mineral usage by plants.

5.
Enhanced nutritional value of food, e.g. Golden rice and sweet potato.

6.
Used to create tailor made plants to supply alternative sources to
industries in the form of starches, fuels and pharmaceuticals.

7.
Production of pest
-
resistant plants, e.g. Bt
-
cotton, Bt
-
corn, rice and
soyabean

etc.




PEST
-

RESISTANT PLANTS


Several nematodes parasitise a wide variety of plants and animals
including human beings.

1.
A nematode
Meloidegyne incognitia

infects the roots of tobacco plants and causes a
great reduction in yield.

2.
A novel strategy was adopted to prevent this infestation which was based on the
process of RNA interference (
RNAi
).
RNAi

takes place in all eukaryotic organisms
as a method of cellular defence. This method involves silencing of a specific mRNA
due to a complementary
dsRNA

molecule that binds to and prevents translation
of the mRNA (silencing).

3.
The source of this complementary RNA could be from an infection by viruses
having RNA genomes or mobile genetic elements (transposons) that replicate via
an RNA intermediate.

4.
Using
Agrobacterium vectors,
nematode
-
specific genes were introduced into the
host plant .

5.
The introduction of DNA was such that it produced both sense and anti
-
sense
RNA in the host cells.

6.
These two RNA’s being complementary to each other formed a double stranded
(
dsRNA
) that initiated
RNAi

and thus, silenced the specific mRNA of the
nematode.

7.
The consequence was that the parasite could not survive in a transgenic host
expressing specific interfering RNA.

8.
The transgenic plant therefore got itself protected from the parasite


Gene therapy is a collection of methods that allow correction of a gene defect
that has been diagnosed in a child/embryo required a mechanism to switch
off a defective gene and to substitute a healthy gene copy.


The first clinical gene therapy was given in 1990 to a 4
-
year old girl with
adenosine deaminase (ADA) deficiency. This enzyme is crucial for


the immune system to function.


The disorder is caused due to the deletion of the gene for adenosine
deaminase.


In some children ADA deficiency can be cured by bone marrow
transplantation; in others it can be treated by enzyme replacement therapy,
in which functional ADA is given to the patient by injection.


As a first step towards gene therapy, lymphocytes from the blood of the
patient are grown in a culture outside the body.


A functional ADA cDNA (using a retroviral vector) is then introduced into
these lymphocytes, which are subsequently returned to the patient.
However, as these cells are not immortal, the patient requires periodic
infusion of such genetically engineered lymphocytes.



However, if the gene isolate from marrow cells producing ADA is introduced
into cells at early embryonic stages, it could be a permanent cure.

MOLECULAR DIAGNOSIS




Recombinant DNA Technology, polymerase chain reaction
(PCR), and Enzyme
-
linked
Immunosorbent

Assay (ELISA) are
some of techniques diagnosis because :


1.
Very low concentration of bacteria or virus (before appearance of visible
symptoms of disease) can be detected by amplification of their nucleic acids.

2.
PCR is now used to detect HIV in suspected AIDS patients.

3.
A probe is a piece of single stranded DNA that is tagged with a radioactive
molecule and it is used to find it complementary by hybridization.

4.
Presence of normal or mutated gene can be detected by this method .

TRANSGENIC ANIMALS


Transgenic animals are those that have had their DNA manipulated to
posses and express an extra or foreign gene, example transgenic rats, rabbits,
pigs, sheep, cow, etc.






Reasons for
genetical

modification of animals


1.
Normal physiology and development


2.
Study of disease


3.
Biological products


4.
Vaccine safety


5.
Chemical safety testing

ETHICAL ISSUES

BIOPIRACY


Biopiracy

is

the

term

used

to

refer

to

the

use

of

bioresources

by

MNCs

and

other

organizations

without

proper

authorization

from

the

country

and

people

concerned

without

compensatory

payment
.



(
i
) Financially rich industrialized nations but poor in biodiversity
exploit the bioresources and traditional knowledge of developing and
underdeveloped poor countries rich in biodiversity.


(ii) Some nations are developing laws to prevent such unauthorized
exploitation of their bioresources and traditional knowledge.

SOME WONDERFUL FACTS

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wit h

human

ear