Biotechnology: Principles and Processes [Courtesy - BANK OF BIOLOGY]
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Sep 28, 2020
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About This Presentation
This PPT would help you to revise and understand the concepts of the lesson BIOTECHNOLOGY: PRINCIPLES AND PROCESSES of class 12.
COURTESY: BANK OF BIOLOGY
This PPT is not posted in any intention, it is posted only for helping the students for revising and preparing this chapter.
Slide Content
What is Biotechnology?
cts and processes useful tc
s or their
) numans.
cts and processes useful tc
s or their
) numans.
hnology
as ‘the integration of natural science and
Toke organisms, cells, parts thereof, and molecular analogues
BIOTECHNOLOGY for products and services’.
as ‘the integration of natural science and
Toke organisms, cells, parts thereof, and molecular analogues
BIOTECHNOLOGY for products and services’.
What is Biotechnology?
In vitro fertilization
(‘test-tube’ baby programme)
Biotechn:
ology
d =
with
www.bankofbiology.com
In vitro fertilization
(‘test-tube’ baby programme)
Biotechn:
ology
d =
with
www.bankofbiology.com
Principles of Biotechnology
Principles of Biotechnology
Bacterial DNA Plasmids
TO) o:
2. Introduction of the identified O O
DNA into the host
+ A vector DNA such as plasmid is used . £
to deliver an alien piece of DNA into Plasmid
the host organism.
Transformed
Cell
E. coli
Host Cell
www.bankofbiology.com
Bacterial DNA Plasmids
TO) o:
2. Introduction of the identified O O
DNA into the host
+ A vector DNA such as plasmid is used . £
to deliver an alien piece of DNA into Plasmid
the host organism.
Transformed
Cell
E. coli
Host Cell
www.bankofbiology.com
Principles of Biotechnology
Basic steos in genetically snocdifying an organisın
+ A piece of alien DNA has no the sequence called Origin of
replication (ori) needed for starting replication. So, it
cannot multiply itself in the progeny cells of the organism.
+ Hence alien DNA is integrated into the recipient genome
(it has ori). It multiplies €: inherits along with host DNA.
Basic steos in genetically snocdifying an organisın
+ A piece of alien DNA has no the sequence called Origin of
replication (ori) needed for starting replication. So, it
cannot multiply itself in the progeny cells of the organism.
+ Hence alien DNA is integrated into the recipient genome
(it has ori). It multiplies €: inherits along with host DNA.
TOOLS OF RECOMBINANT DNA TECHNOLOGY
TOOLS OF RECOMBINANT DNA TECHNOLOGY
Ampicillin Tetracycline
Resistance a Resistance
ie UN O
Re bi: rt
a ee sas an =
(4363 bp) Host Cell
NS
Transformed
Cell
Cloning,
Vectors
..
L scissors’) 4
www.bankofbiology.com
Ampicillin Tetracycline
Resistance a Resistance
ie UN O
Re bi: rt
a ee sas an =
(4363 bp) Host Cell
NS
Transformed
Cell
Cloning,
Vectors
..
L scissors’) 4
www.bankofbiology.com
TOOLS OF RECOMBINANT DNA TECHNOLOGY
+ These are the enzymes which cut DNA
at specific sites into fragments.
+ They belong to a class of enzymes
called
+ These are the enzymes which cut DNA
at specific sites into fragments.
+ They belong to a class of enzymes
called
TOOLS OF RECOMBINA NOLOGY
E
1. Restriction Enzymes (“molecular scissors”)
+ In 1963, two enzymes responsible for
restricting growth of bacteriophage in E. ~ L/
coli were isolated. One enzyme added
methyl groups to DNA. The other
Ecorı
(restriction endonuclease) cut DNA. +
— os 61. — 2:
on C Y 1 A AG a 5
More than 900 restriction enzymes have
been isolated from over 230 strains of
bacteria.
Cleavage | pus
Sticky ends
G, RATTC a
c
5
vun CT TAA cn 5
www.bankofbiology.com
E
1. Restriction Enzymes (“molecular scissors”)
+ In 1963, two enzymes responsible for
restricting growth of bacteriophage in E. ~ L/
coli were isolated. One enzyme added
methyl groups to DNA. The other
Ecorı
(restriction endonuclease) cut DNA. +
— os 61. — 2:
on C Y 1 A AG a 5
More than 900 restriction enzymes have
been isolated from over 230 strains of
bacteria.
Cleavage | pus
Sticky ends
G, RATTC a
c
5
vun CT TAA cn 5
www.bankofbiology.com
TOOLS OF RECOMBINANT DNA TECHNOLOGY
1. Restriction Enzymes _ [iNaimingfofiRestrictionjenzymes)
« First letter indicates genus.
* Second two letters indicate species
of the prokaryotic cell from which
they were isolated. >
» E.g. EcoRI comes from E. coli RY 13. Roman number = the order in
which enzymes were isolated
from that strain of bacteria.
www.bankofbiology.com
1. Restriction Enzymes _ [iNaimingfofiRestrictionjenzymes)
« First letter indicates genus.
* Second two letters indicate species
of the prokaryotic cell from which
they were isolated. >
» E.g. EcoRI comes from E. coli RY 13. Roman number = the order in
which enzymes were isolated
from that strain of bacteria.
www.bankofbiology.com
OOLS OF RECOMBINANT DNA TECHNOLOGY
on Enzymes
“The enzyme cuts both DNA Ecol cuts the DNA between bases
Exonucleases rares of the eue sits Gand À only when tne soquence
ERES O present in the DNA
They remove nucleotides from Vector DNA Poreiga DNA
the ends of the DNA. aa arr D NO
Endonucleases | j
They cut at specific positions Sticky end
within DNA. DNA
Sticky end
DNA fragments join at sticky ends
They bind to specific recognition
sequence of the DNA and cut the
two strands at specific points. pobla
www.bankofbiology.com
on Enzymes
“The enzyme cuts both DNA Ecol cuts the DNA between bases
Exonucleases rares of the eue sits Gand À only when tne soquence
ERES O present in the DNA
They remove nucleotides from Vector DNA Poreiga DNA
the ends of the DNA. aa arr D NO
Endonucleases | j
They cut at specific positions Sticky end
within DNA. DNA
Sticky end
DNA fragments join at sticky ends
They bind to specific recognition
sequence of the DNA and cut the
two strands at specific points. pobla
www.bankofbiology.com
OF RECOMBINANT DNA TECHNOLOGY
S |
» First restriction endonuclease is Atut 5: 1113 G6 all: 3°
adi m oia
» It cuts DNA by recognizing a specific u © dea rec... 3
4 a nee ff w 696...
sequence of six base pairs. This is
. Hindi a n'es Cr To 3
called the recognition sequence for 3 add <7 IE
Hind ll. Ecort 3: [IE ta Age... 3
Hind Il Alu and Haelll produce blunt enas
5 GTPy Pu AC 3: BamHl Hindill and EcoRI produce “sticky” ends
3...CAPu[PyTG...5' Recognition sites of various types of
restriction endonucleases
www.bankofbiology.com
S |
» First restriction endonuclease is Atut 5: 1113 G6 all: 3°
adi m oia
» It cuts DNA by recognizing a specific u © dea rec... 3
4 a nee ff w 696...
sequence of six base pairs. This is
. Hindi a n'es Cr To 3
called the recognition sequence for 3 add <7 IE
Hind ll. Ecort 3: [IE ta Age... 3
Hind Il Alu and Haelll produce blunt enas
5 GTPy Pu AC 3: BamHl Hindill and EcoRI produce “sticky” ends
3...CAPu[PyTG...5' Recognition sites of various types of
restriction endonucleases
www.bankofbiology.com
OLS OF RECOMBINANT DNA TECHNOLOGY
1. Restriction Enzymes
* Restriction endonuclease recognizes At 3° 1117 Gye alll 3°
a specific palindromic nucleotide — 3: oe cas si 5
sequences. It is a sequence of base sam 5 MKaree...s
i CTW Gye...
pairs that read the same on two
ne) ‘ in 3" 1 Hinge IIA OS
strands in 5' >3'andin3' > 5 me :
irections. < SiS
direct EcoRl 53 :
Alul and Haelll produce blunt ends
BamHI Hindill and EcoRI produce “sticky” ends
Recognition sites of various types of
restriction endonucleases
www.bankofbiology.com
1. Restriction Enzymes
* Restriction endonuclease recognizes At 3° 1117 Gye alll 3°
a specific palindromic nucleotide — 3: oe cas si 5
sequences. It is a sequence of base sam 5 MKaree...s
i CTW Gye...
pairs that read the same on two
ne) ‘ in 3" 1 Hinge IIA OS
strands in 5' >3'andin3' > 5 me :
irections. < SiS
direct EcoRl 53 :
Alul and Haelll produce blunt ends
BamHI Hindill and EcoRI produce “sticky” ends
Recognition sites of various types of
restriction endonucleases
www.bankofbiology.com
es (‘mol
Pre ensyne outa both
* Restriction enzymes cut the
strand a little away from the
Vector DNA
strands at the same site
ecular
DNA
y
centre of palindrome sites,
but between the same two
bases on the opposite
strands. This leaves single
stranded overhanging
stretches at the ends. They
are called sticky ends. I NONI NG
www.bankofbiology.com
“Sticky end
DNA fragments join at sticky ends
|
IAA
Recombinant DNA
im
|
DNA TECHNOLOGY
EcoRI cuts the DNA between bases
G and A only when the sequence
GAATTC is present in the DNA
Foreign DNA
Sticky end
Pre ensyne outa both
* Restriction enzymes cut the
strand a little away from the
Vector DNA
strands at the same site
ecular
DNA
y
centre of palindrome sites,
but between the same two
bases on the opposite
strands. This leaves single
stranded overhanging
stretches at the ends. They
are called sticky ends. I NONI NG
www.bankofbiology.com
“Sticky end
DNA fragments join at sticky ends
|
IAA
Recombinant DNA
im
|
DNA TECHNOLOGY
EcoRI cuts the DNA between bases
G and A only when the sequence
GAATTC is present in the DNA
Foreign DNA
Sticky end
es (‘molecular
“The enzyme cuts both
» Sticky ends form H-bonds with
their complementary cut
Vector DNA
strands at the same site
DNA
y
counterparts. This stickiness
facilitates action of DNA ligase.
When cut by the same
restriction enzyme, the DNA
fragments have the same kind
of sticky-ends. They are joined
by DNA ligases. RIVIS
www.bankofbiology.com
“Sticky end
DNA fragments join at sticky ends
|
IAA
Recombinant DNA
im
|
DNA TECHNOLOGY
EcoRI cuts the DNA between bases
G and A only when the sequence
GAATTC is present in the DNA
Foreign DNA
Sticky end
“The enzyme cuts both
» Sticky ends form H-bonds with
their complementary cut
Vector DNA
strands at the same site
DNA
y
counterparts. This stickiness
facilitates action of DNA ligase.
When cut by the same
restriction enzyme, the DNA
fragments have the same kind
of sticky-ends. They are joined
by DNA ligases. RIVIS
www.bankofbiology.com
“Sticky end
DNA fragments join at sticky ends
|
IAA
Recombinant DNA
im
|
DNA TECHNOLOGY
EcoRI cuts the DNA between bases
G and A only when the sequence
GAATTC is present in the DNA
Foreign DNA
Sticky end
» Sticky ends form H-bonds with
their complementary cut „— DNA and vector DNA st epociflo point à
counterparts. This stickiness cy
> E E >00 20 20000
facilitates action of DIVA ligase. Ligases join foreign
Past
When cut by the same
restriction enzyme, the DNA representation Recnesitenst DNA
recombinant
fragments have the same kind DNA technology
of sticky-ends. They are joined
by DNA ligases.
E.coli
(Cloning host)
Sow EB
www.bankofbiology.com
their complementary cut „— DNA and vector DNA st epociflo point à
counterparts. This stickiness cy
> E E >00 20 20000
facilitates action of DIVA ligase. Ligases join foreign
Past
When cut by the same
restriction enzyme, the DNA representation Recnesitenst DNA
recombinant
fragments have the same kind DNA technology
of sticky-ends. They are joined
by DNA ligases.
E.coli
(Cloning host)
Sow EB
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TOOLS OF RECOMBINANT DNA TECHNOLOGY
2, Cloning Vactors |
+ Itis a DNA molecule that can carry a foreign DNA segment and replicate
inside the host cells.
°NE:p:
Bacterial DNA Plasmids
2, Cloning Vactors |
+ Itis a DNA molecule that can carry a foreign DNA segment and replicate
inside the host cells.
°NE:p:
Bacterial DNA Plasmids
TOOLS OF RECOMBINANT DNA TECHNOLOGY
2, Cloning Vactors bankofbiology.com |
are autonomously replicating circular extra-chromosomal DNA of
bacteria. Some plasmids have only 1-2 copies per cell. Others have 15-100
copies per cell.
(high number per cell) have very high copy numbers of their
genome within the bacterial cells.
Bacterial DNA Plasmids
2, Cloning Vactors bankofbiology.com |
are autonomously replicating circular extra-chromosomal DNA of
bacteria. Some plasmids have only 1-2 copies per cell. Others have 15-100
copies per cell.
(high number per cell) have very high copy numbers of their
genome within the bacterial cells.
Bacterial DNA Plasmids
TOOLS OF RECOMBINANT DNA TECHNOLOGY
2, Cloning Vactors |
« When the cloning vectors are multiplied in the host, the linked piece of DNA
is also multiplied to the numbers equal to the copy number of the vectors.
Bacterial DNA Plasmids
2, Cloning Vactors |
« When the cloning vectors are multiplied in the host, the linked piece of DNA
is also multiplied to the numbers equal to the copy number of the vectors.
Bacterial DNA Plasmids
TOOLS OF RECOMBINANT DNA TECHNOLOGY
Features required for cloning into a vector
EcoR 1-—Cla L__Hind Il
Origin of replication (ori)
Selectable marker
(Marker gene)
Cloning sites
. Vectors for cloning genes
in plants & animals
E.coli cloning vector pBR322
www.bankofbiology.com
Features required for cloning into a vector
EcoR 1-—Cla L__Hind Il
Origin of replication (ori)
Selectable marker
(Marker gene)
Cloning sites
. Vectors for cloning genes
in plants & animals
E.coli cloning vector pBR322
www.bankofbiology.com
OF RECOMBINANT DNA TEC OG
bankofbiology.com
Features required for cloning into a vector
Pvu | a
+ It is a sequence where replication starts. Poth u cs
amp’ te
* A piece of DNA linked to ori can replicate within “
| pBR322 Sal 1
the host cells. a
+ It also controls the copy number of linked DNA. So, aa _ top 4
for getting many copies of target DNA, it should be -
cloned in a vector whose origin support high copy Pvu I
number. E.coli cloning vector pBR322
iology.com
bankofbiology.com
Features required for cloning into a vector
Pvu | a
+ It is a sequence where replication starts. Poth u cs
amp’ te
* A piece of DNA linked to ori can replicate within “
| pBR322 Sal 1
the host cells. a
+ It also controls the copy number of linked DNA. So, aa _ top 4
for getting many copies of target DNA, it should be -
cloned in a vector whose origin support high copy Pvu I
number. E.coli cloning vector pBR322
iology.com
OOLS OF RECOMBINANT DNA TE O
Features required for cloning into a vector
Cial, Mind un
Damit
cloning bj
* It is a gene that helps to select transformants and vector = Le
eliminate the non-transformants. ppR322 a I
* If a piece of DNA is introduced in a host bacterium,
it is called transformation. Such bacterium is Q> >
Recombinant
transformant.
+ If transformation does not take place, it is non-
transformant. Transformed
Host Cell
www.bankofbiology.com
Features required for cloning into a vector
Cial, Mind un
Damit
cloning bj
* It is a gene that helps to select transformants and vector = Le
eliminate the non-transformants. ppR322 a I
* If a piece of DNA is introduced in a host bacterium,
it is called transformation. Such bacterium is Q> >
Recombinant
transformant.
+ If transformation does not take place, it is non-
transformant. Transformed
Host Cell
www.bankofbiology.com
TOOLS OF RECOMBINANT DNA TECHNOLOGY
2. Cloning F
Vectors
B. Selectable marker (marker gene) Pa
cloning
vector
pBR322
eatures required for cloning into a vecto
* Selectable markers of E. coli include genes
encoding resistance to antibiotics like ampicillin,
chloramphenicol, tetracycline, kanamycin, etc.
+ Normal E. coli cells have no resistance against
these antibiotics.
www.bankofbiology.com
2. Cloning F
Vectors
B. Selectable marker (marker gene) Pa
cloning
vector
pBR322
eatures required for cloning into a vecto
* Selectable markers of E. coli include genes
encoding resistance to antibiotics like ampicillin,
chloramphenicol, tetracycline, kanamycin, etc.
+ Normal E. coli cells have no resistance against
these antibiotics.
www.bankofbiology.com
TOOLS OF RECOMBINANT DNA TECHNOLOGY
bankofbiology.com
Features required for cloning into a vector
a Cla L__Hind III
EeoR I— If
Pvul
* To link the alien DNA, the vector needs a Paths
A /
single or very few for oa (ABER
restriction enzymes. \
+ More than one recognition sites generates «x: mp y
several fragments. It complicates the gene 7 \
cloning. Pvu II
E. Coli cloning vector pBR322
D:
amp“ tet"
w.bankofbiology.com
bankofbiology.com
Features required for cloning into a vector
a Cla L__Hind III
EeoR I— If
Pvul
* To link the alien DNA, the vector needs a Paths
A /
single or very few for oa (ABER
restriction enzymes. \
+ More than one recognition sites generates «x: mp y
several fragments. It complicates the gene 7 \
cloning. Pvu II
E. Coli cloning vector pBR322
D:
amp“ tet"
w.bankofbiology.com
OLS OF RECOMBINANT DNA TEC OGY
Features required for cloning into a vector
Cla I__Hind Ill
EcoR 1 I
* Ligation of alien DNA is carried out at a
restriction site present in one of the two
antibiotic resistance genes.
* E.g. ligation of a foreign DNA at Bam HI site of
tetracycline resistance gene in vector pBR322.
As a result, recombinant plasmid is formed.
* If ligation does not occur, it is called non-
recombinant plasmid.
www.bankofbiology.com
Pvu II
E. Coli cloning vector pBR322
Features required for cloning into a vector
Cla I__Hind Ill
EcoR 1 I
* Ligation of alien DNA is carried out at a
restriction site present in one of the two
antibiotic resistance genes.
* E.g. ligation of a foreign DNA at Bam HI site of
tetracycline resistance gene in vector pBR322.
As a result, recombinant plasmid is formed.
* If ligation does not occur, it is called non-
recombinant plasmid.
www.bankofbiology.com
Pvu II
E. Coli cloning vector pBR322
TOOLS OF RECOMBINANT DNA TEC LOGY
* When a foreign DNA is inserted within a gene
of bacteria, that gene is inactivated. It is called
insertional inactivation. Here, the recombinant
plasmids lose tetracycline resistance due to
insertion of foreign DNA.
+ When the plasmids are introduced into E. coli
cells, 3 types of cells are obtained:
www.bankofbiology.com
Features required for cloning into a vector
Transformed | Non-transformed
cell y cell
N Nutrient medium
+
ser Ampicillin
Overnight
growth
el
Ampicillin-resistant colonies
* When a foreign DNA is inserted within a gene
of bacteria, that gene is inactivated. It is called
insertional inactivation. Here, the recombinant
plasmids lose tetracycline resistance due to
insertion of foreign DNA.
+ When the plasmids are introduced into E. coli
cells, 3 types of cells are obtained:
www.bankofbiology.com
Features required for cloning into a vector
Transformed | Non-transformed
cell y cell
N Nutrient medium
+
ser Ampicillin
Overnight
growth
el
Ampicillin-resistant colonies
OLS OF RECOMBINANT DNA TEC LOGY
Features required for cloning into a vector
1. Non-transformants: They have no plasmid. So errs
>
they are not resistant to either tetracycline or
ampicillin.
. Transformants with non-recombinant plasmid:
They are resistant to both tetracycline & CS) xD)
ampicillin. Transformed with
. Transformants with recombinant plasmid: They Non transformed rl
vector
are resistant only to ampicillin.
www.bankofbiology.com
Features required for cloning into a vector
1. Non-transformants: They have no plasmid. So errs
>
they are not resistant to either tetracycline or
ampicillin.
. Transformants with non-recombinant plasmid:
They are resistant to both tetracycline & CS) xD)
ampicillin. Transformed with
. Transformants with recombinant plasmid: They Non transformed rl
vector
are resistant only to ampicillin.
www.bankofbiology.com
OOLS OF RECOMBINANT DNA TEC GY
Features required for cloning into a vector
(> tat cane inactive
¡DNA F (recombinant)
Na? Le tet gene active
* Recombinant plasmids can be selected out from PNA horrecomoheng
non-recombinant ones by plating the transformants E.colicells | Transformation
=> | plate on ampicillin
containing medium Recombinant
on ampicillin medium. Then the transformants are
transferred on tetracycline medium.
* The recombinants grow in ampicillin medium but ñ
A 4 E epica plating
not on tetracycline medium. But, non-recombinants A
grow on medium containing both the antibiotics. medium
Master plate Replica plate
Bacterial
colony Non-recombinant |
www.bankofbiology.com
Features required for cloning into a vector
(> tat cane inactive
¡DNA F (recombinant)
Na? Le tet gene active
* Recombinant plasmids can be selected out from PNA horrecomoheng
non-recombinant ones by plating the transformants E.colicells | Transformation
=> | plate on ampicillin
containing medium Recombinant
on ampicillin medium. Then the transformants are
transferred on tetracycline medium.
* The recombinants grow in ampicillin medium but ñ
A 4 E epica plating
not on tetracycline medium. But, non-recombinants A
grow on medium containing both the antibiotics. medium
Master plate Replica plate
Bacterial
colony Non-recombinant |
www.bankofbiology.com
OLS OF RECOMBINANT DNA TEC LOGY
Features required for cloning into a vector
Ls tet gene inactive
FE "DNA F (recombinant)
7 Ce tet gene active
Æ-rDNA Y (non-recombinant)
+ Thus, one antibiotic resistance gene helps to Boolean Trnstomasen,
select the transformants. oak Recombinant
. a “Le . . Non-+ bi it
* The inactivated antibiotic resistance gene ne A
helps to select recombinants. (33 Hevea patos
on tetracycline
medium
Master plate Replica plate
www.bankofbiology.com
Features required for cloning into a vector
Ls tet gene inactive
FE "DNA F (recombinant)
7 Ce tet gene active
Æ-rDNA Y (non-recombinant)
+ Thus, one antibiotic resistance gene helps to Boolean Trnstomasen,
select the transformants. oak Recombinant
. a “Le . . Non-+ bi it
* The inactivated antibiotic resistance gene ne A
helps to select recombinants. (33 Hevea patos
on tetracycline
medium
Master plate Replica plate
www.bankofbiology.com
TOOLS OF RECOMBINANT DNA TEC OGY
bankofbiology.com
Features required for cloning into a vector
a
laszAMiS
Competent E. coli
ir
: : A - — > —
* But this type of selection of recombinants is a Foreign DNA
A D . insert within lacZ
difficult procedure because it needs simultaneous + ds + chromogenic
Lara
plating on 2 plates having different antibiotics. 4
White eier
+ So, alternative selectable markers have Plasmid O—
meee vector L,
developed based on their ability to produce
colour in presence of a chromogenic substrate. me en
Blue colonies
www.bankofbiology.com
bankofbiology.com
Features required for cloning into a vector
a
laszAMiS
Competent E. coli
ir
: : A - — > —
* But this type of selection of recombinants is a Foreign DNA
A D . insert within lacZ
difficult procedure because it needs simultaneous + ds + chromogenic
Lara
plating on 2 plates having different antibiotics. 4
White eier
+ So, alternative selectable markers have Plasmid O—
meee vector L,
developed based on their ability to produce
colour in presence of a chromogenic substrate. me en
Blue colonies
www.bankofbiology.com
GY
Features required for cloning into a vector
+ In this, a recombinant DNA is inserted into the
— >
coding sequence (gene) of an enzyme, /- Foreign DNA er
galactosidase. So the gene is inactivated (insertional + substrate
4
White colonies
inactivation). Such colonies do not produce colour.
These are identified as recombinant colonies. Plasmid
If the plasmid in bacteria have no an insert, it gives sia e O (O e)
blue coloured colonies in presence of chromogenic no insert + chromogenic
substrate
substrate. 4
Blue colonies
www.bankofbiology.com
Features required for cloning into a vector
+ In this, a recombinant DNA is inserted into the
— >
coding sequence (gene) of an enzyme, /- Foreign DNA er
galactosidase. So the gene is inactivated (insertional + substrate
4
White colonies
inactivation). Such colonies do not produce colour.
These are identified as recombinant colonies. Plasmid
If the plasmid in bacteria have no an insert, it gives sia e O (O e)
blue coloured colonies in presence of chromogenic no insert + chromogenic
substrate
substrate. 4
Blue colonies
www.bankofbiology.com
OLS OF RECOMBINANT DNA TECHNOLOGY
Features required for cloning into a vector
+ In this, a recombinant DNA is inserted into the
coding sequence (gene) of an enzyme,
So the gene is inactivated
Such colonies do not produce colour.
These are identified as recombinant colonies.
* If the plasmid in bacteria have no an insert, it \ ,
colonies in presence of chromogenic TES
un
substrate.
Blue colonies and white colonies
Features required for cloning into a vector
+ In this, a recombinant DNA is inserted into the
coding sequence (gene) of an enzyme,
So the gene is inactivated
Such colonies do not produce colour.
These are identified as recombinant colonies.
* If the plasmid in bacteria have no an insert, it \ ,
colonies in presence of chromogenic TES
un
substrate.
Blue colonies and white colonies
TOOLS OF RECOMBINANT DNA TECHNOLOGY
Se Features required for cloning into a vector
* Genetic tools of some pathogens is
transformed into useful vectors for
delivering genes to plants & animals. E.g.
(a pathogen
of many dicot plants).
Se Features required for cloning into a vector
* Genetic tools of some pathogens is
transformed into useful vectors for
delivering genes to plants & animals. E.g.
(a pathogen
of many dicot plants).
OLS OF RECOMBINANT DNA TEC OG
Features required for clonin
into a vector
> Agrobacterium tumefaciens can deliver a piece of
DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals
required by the pathogen.
Tumor inducing (Ti) plasmid of A. tumefaciens is
modified into a cloning vector whi
pathogenic but can use the mechanisms to deliver
genes of interest into plants.
www.bankofbiology.com
Features required for clonin
into a vector
> Agrobacterium tumefaciens can deliver a piece of
DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals
required by the pathogen.
Tumor inducing (Ti) plasmid of A. tumefaciens is
modified into a cloning vector whi
pathogenic but can use the mechanisms to deliver
genes of interest into plants.
www.bankofbiology.com
OLS OF RECOMBINANT DNA TEC
bankofbiology.com
Features required for cloning into a vector
> Agrobacterium tumefaciens can deliver a piece of
DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals
required by the pathogen.
Tumor inducing (Ti) plasmid of A. tumefaciens is
modified into a cloning vector whi
pathogenic but can use the mechanisms to deliver
genes of interest into plants.
www.bankofbiology.com
bankofbiology.com
Features required for cloning into a vector
> Agrobacterium tumefaciens can deliver a piece of
DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals
required by the pathogen.
Tumor inducing (Ti) plasmid of A. tumefaciens is
modified into a cloning vector whi
pathogenic but can use the mechanisms to deliver
genes of interest into plants.
www.bankofbiology.com
TOOLS OF RECOMBINANT DNA TEC OGY
2. Cloning F
eatures required for cloning into a vector
Vectors
Infection of retrovirus
proto-oncogene
®
—
> Retroviruses in animals can transform
normal cells into cancerous cells. mela cancerous cl
> So they are used to deliver desirable genes
into animal cells.
www.bankofbiology.com
2. Cloning F
eatures required for cloning into a vector
Vectors
Infection of retrovirus
proto-oncogene
®
—
> Retroviruses in animals can transform
normal cells into cancerous cells. mela cancerous cl
> So they are used to deliver desirable genes
into animal cells.
www.bankofbiology.com
OOLS OF RECOMBINANT DNA TEC
3, Cornostant Host (For Transformation with Re
cat watt =
2 = a . bacterial Non-transformed
* Since DNA is a hydrophilic molecule, it genomic DNA bacteria
cannot pass through cell membranes. id
So the bacterial cells must be made FRE
‘competent’ to take up alien DNA or
plasmid. Competent cell
For this, bacterial cells are treated with a
specific concentration of a divalent
cation (e.g. calcium). So DNA enters the
bacterium through pores in cell wall.
Plasmid transported
into cell
www.bankofbiology.com
3, Cornostant Host (For Transformation with Re
cat watt =
2 = a . bacterial Non-transformed
* Since DNA is a hydrophilic molecule, it genomic DNA bacteria
cannot pass through cell membranes. id
So the bacterial cells must be made FRE
‘competent’ to take up alien DNA or
plasmid. Competent cell
For this, bacterial cells are treated with a
specific concentration of a divalent
cation (e.g. calcium). So DNA enters the
bacterium through pores in cell wall.
Plasmid transported
into cell
www.bankofbiology.com
OF RECOMBINANT DNA TEC LOGY
tant Host (For Transformation with Racsornoinant DNA) |
= bankofbiology.com
ell wall =
acterial Non-transformed
genomic DNA bacteria
id
* Such cells are incubated with
recombinant DNA on ice.
* Then they are placed briefly at 42°C (heat Competent cell
shock) and put them back on ice.
+ This enables the bacteria to take up the
recombinant DNA.
Plasmid transported
into cell
www.bankofbiology.com
tant Host (For Transformation with Racsornoinant DNA) |
= bankofbiology.com
ell wall =
acterial Non-transformed
genomic DNA bacteria
id
* Such cells are incubated with
recombinant DNA on ice.
* Then they are placed briefly at 42°C (heat Competent cell
shock) and put them back on ice.
+ This enables the bacteria to take up the
recombinant DNA.
Plasmid transported
into cell
www.bankofbiology.com
TOOLS OF RECOMBINA
Cornostars y EIER DS NS OSEO WIEN 15590055 FS LADS. |
Other methods to introduce alien DNA into host cells
‘orien ona o NE
mn “
‘crept
E Gene gun
Micro-injection: Recombinant DNA is directly injected into nucleus of an animal cell.
Biolistics (gene gun): Cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA. This method is suitable for plants.
‘Disarmed pathogen’ vectors: When it infects the cell, transfer recombinant DNA into
the host.
www.bankofbiology.com
Cornostars y EIER DS NS OSEO WIEN 15590055 FS LADS. |
Other methods to introduce alien DNA into host cells
‘orien ona o NE
mn “
‘crept
E Gene gun
Micro-injection: Recombinant DNA is directly injected into nucleus of an animal cell.
Biolistics (gene gun): Cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA. This method is suitable for plants.
‘Disarmed pathogen’ vectors: When it infects the cell, transfer recombinant DNA into
the host.
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
of Recombinant DNA into Host Cell
5. Obtaining the Foreign Gene Product
| 6. Downstream Processing
www.bankofbiology.com
of Recombinant DNA into Host Cell
5. Obtaining the Foreign Gene Product
| 6. Downstream Processing
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
al, lolo Gf tna Canale Marartel (DIVA) )
za
bankofbiology.com
>
+» Treat bacterial cells/plant or animal
tissue with enzymes like lysozyme
(bacteria), cellulase (plants), chitinase
(fungus) etc.
* The cell is broken releasing DNA & other
macromolecules (RNA, proteins,
polysaccharides & lipids).
www.bankofbiology.com
al, lolo Gf tna Canale Marartel (DIVA) )
za
bankofbiology.com
>
+» Treat bacterial cells/plant or animal
tissue with enzymes like lysozyme
(bacteria), cellulase (plants), chitinase
(fungus) etc.
* The cell is broken releasing DNA & other
macromolecules (RNA, proteins,
polysaccharides & lipids).
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
| 1. sdktion of dhe Gane Metal (DNA)
e. Proteins are removed by treating
+ RNA is removed by treating with rib
with protease. Other molecules are removed by appropriate treatments.
ipitates out as fine threads in
+ When chi tha is added, purified
the suspension.
ASA alcohol onto the sides Spooting of
until two distinct layers are genetic material
observed
separated
sail a
Separated DNA can be removed by spooling
www.bankofbiology.com
| 1. sdktion of dhe Gane Metal (DNA)
e. Proteins are removed by treating
+ RNA is removed by treating with rib
with protease. Other molecules are removed by appropriate treatments.
ipitates out as fine threads in
+ When chi tha is added, purified
the suspension.
ASA alcohol onto the sides Spooting of
until two distinct layers are genetic material
observed
separated
sail a
Separated DNA can be removed by spooling
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
2, Gating of DNA et Speciits Loctiions }
* Purified DNA is incubated with the restriction enzyme. As a result, DNA digests.
+ DNA fragments are separated by a technique called gel electrophoresis.
+ Agarose gel electrophoresis is employed to check the progression of a restriction
enzyme E
Wells
AT bands a
res 77
En
Gel electrophoresis showing migration of
undigested (lane 1) and digested set of DNA
q fragments (lane 2 to 4)
www.bankofbiology.com
2, Gating of DNA et Speciits Loctiions }
* Purified DNA is incubated with the restriction enzyme. As a result, DNA digests.
+ DNA fragments are separated by a technique called gel electrophoresis.
+ Agarose gel electrophoresis is employed to check the progression of a restriction
enzyme E
Wells
AT bands a
res 77
En
Gel electrophoresis showing migration of
undigested (lane 1) and digested set of DNA
q fragments (lane 2 to 4)
www.bankofbiology.com
PROCESSES OF RECOMBINAN
RACUttinelo DNA et Saadiile Locations
+ DNA is negatively charged. So it moves towards the anode.
+ DNA fragments are separated according to their size through sieving effect of agarose
gel (polymer from sea weeds). Smaller sized fragment move farther.
+ The process is repeated with the vector DNA also.
Wells
START WELLS FILLED
DNA WITH SAMPLE
> LS
Largest Smallest om =
AA /
Gel electrophoresis showing migration of
undigested (lane 1) and digested set of DNA +
==;
rr
fragments (lane 2 to 4)
www.bankofbiology.com
RACUttinelo DNA et Saadiile Locations
+ DNA is negatively charged. So it moves towards the anode.
+ DNA fragments are separated according to their size through sieving effect of agarose
gel (polymer from sea weeds). Smaller sized fragment move farther.
+ The process is repeated with the vector DNA also.
Wells
START WELLS FILLED
DNA WITH SAMPLE
> LS
Largest Smallest om =
AA /
Gel electrophoresis showing migration of
undigested (lane 1) and digested set of DNA +
==;
rr
fragments (lane 2 to 4)
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNO
2, ug O DNA el Spactiie locations )
+ DNA fragments can be seen as bright orange coloured bands when they are
stained with ethidium bromide and exposed to UV radiation.
Wells
DNA —
AT bands a
Largest Smallest
Gel electrophoresis showing migration of
undigested (lane 1) and digested set of DNA
fragments (lane 2 to 4)
wy
2, ug O DNA el Spactiie locations )
+ DNA fragments can be seen as bright orange coloured bands when they are
stained with ethidium bromide and exposed to UV radiation.
Wells
DNA —
AT bands a
Largest Smallest
Gel electrophoresis showing migration of
undigested (lane 1) and digested set of DNA
fragments (lane 2 to 4)
wy
PROCES OF RECOMBINANT DNA TECHNOLOG
2, Cu où DINA ak Spediiis Locations
* DNA bands are cut out from agarose gel. This is called elution.
* The cut-out gene of interest from source DNA and cut vector are mixed and ligase
is added. It creates recombinant DNA.
|. Ho.
bankofbiology.com
Gene of
interest
Recombinant
Ligase
> DNA
and add to Gel
Ucar aad Vector DNA
Transfer to GET Wash and then
Spin Column elute DNA ina
small volume.
www.bankofbiology.com
2, Cu où DINA ak Spediiis Locations
* DNA bands are cut out from agarose gel. This is called elution.
* The cut-out gene of interest from source DNA and cut vector are mixed and ligase
is added. It creates recombinant DNA.
|. Ho.
bankofbiology.com
Gene of
interest
Recombinant
Ligase
> DNA
and add to Gel
Ucar aad Vector DNA
Transfer to GET Wash and then
Spin Column elute DNA ina
small volume.
www.bankofbiology.com
* Polymerase Chain Reaction (PCR) is the
synthesis of multiple copies of the gene
of interest in vitro using 2 sets of primers
& the enzyme DNA polymerase.
+ Primers are small chemically synthesized
oligonucleotides that are
complementary to the regions of DNA.
synthesis of multiple copies of the gene
of interest in vitro using 2 sets of primers
& the enzyme DNA polymerase.
+ Primers are small chemically synthesized
oligonucleotides that are
complementary to the regions of DNA.
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
S Ampliticeriion of Gena of Interest using PER
Region to be amplified
e A AAA Pr Ze: —_
ST 1° «on.
y- , Denaturation 1. Denaturation
q Poner à. Primer Annealing
VRR sd 2. Annealing
TT SSSR 7 1
q = Extension
nl | | .
3. Extension
www.bankofbiology.com
S Ampliticeriion of Gena of Interest using PER
Region to be amplified
e A AAA Pr Ze: —_
ST 1° «on.
y- , Denaturation 1. Denaturation
q Poner à. Primer Annealing
VRR sd 2. Annealing
TT SSSR 7 1
q = Extension
nl | | .
3. Extension
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
S Amplitiiceriion of Gena of Interest using PER
e nn, Steps of PCR
U TT, ss osa
s
4 a _Denaturation
5; Primer. =—_ Annealing
moe + It is the heating of target DNA (gene of
VER | interest) at high temperature (94° C) to
TT O3 nun separate the strands.
+ 4 Extension
„ii | + Each strands act as template for DNA
td a synthesis.
EEEEEEEEEET
mae | a
www.bankofbiology.com
S Amplitiiceriion of Gena of Interest using PER
e nn, Steps of PCR
U TT, ss osa
s
4 a _Denaturation
5; Primer. =—_ Annealing
moe + It is the heating of target DNA (gene of
VER | interest) at high temperature (94° C) to
TT O3 nun separate the strands.
+ 4 Extension
„ii | + Each strands act as template for DNA
td a synthesis.
EEEEEEEEEET
mae | a
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
& Ampliiication of Gana of Intarast using PER
IT TE TT moss
y 8
Heat D. '
A eee o bankofbiology.com
a Primers. © Primer Annealing
DNA polymerase .
VER . + Itisthe joining of the two primers (at
B | 52° C) at the 3’ end of the DNA
4 5 Extension
nl Mu | |. templates.
www.bankofbiology.com
& Ampliiication of Gana of Intarast using PER
IT TE TT moss
y 8
Heat D. '
A eee o bankofbiology.com
a Primers. © Primer Annealing
DNA polymerase .
VER . + Itisthe joining of the two primers (at
B | 52° C) at the 3’ end of the DNA
4 5 Extension
nl Mu | |. templates.
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNO
9S. Amplitieetion of Gane o? Intarast using PER
>, Region to be amplified
> o Steps of PCR 3. Extension
_ Denaturation |. itis the a
MSDE a thermostable DNA polymerase called
aaa
2 y vou
Annealing
t is isolated from a
Becta um, . lt remains
active in high temperature during the
a dani denaturation of double stranded DNA.
Lu, + Through continuous replication the DNA
segment is amplified up to 1
+ The amplified fragment can be used to ligate
with a vector for further cloning.
www.bankofbiology.com
9S. Amplitieetion of Gane o? Intarast using PER
>, Region to be amplified
> o Steps of PCR 3. Extension
_ Denaturation |. itis the a
MSDE a thermostable DNA polymerase called
aaa
2 y vou
Annealing
t is isolated from a
Becta um, . lt remains
active in high temperature during the
a dani denaturation of double stranded DNA.
Lu, + Through continuous replication the DNA
segment is amplified up to 1
+ The amplified fragment can be used to ligate
with a vector for further cloning.
www.bankofbiology.com
OCESSES OF RECOMBINANT DNA TECHNOLO
& Ampoliiicstion of Gana o? Intarast using PER |
PCR COMPONENTS PCR PROCESS
PCR AT A m ce 6]
A T x trands separate
GLANCE ONASempe Pire Nucleotides Lores Comité
Teapobmerese | MixEufer PCR Tube a LG
<i
= J Tir.
a | |
\ VAS 72°C - Synesise new stand 3, Extension
Pe SEBEREEBEN
Thermal Cycler
www.bankofbiology.com
& Ampoliiicstion of Gana o? Intarast using PER |
PCR COMPONENTS PCR PROCESS
PCR AT A m ce 6]
A T x trands separate
GLANCE ONASempe Pire Nucleotides Lores Comité
Teapobmerese | MixEufer PCR Tube a LG
<i
= J Tir.
a | |
\ VAS 72°C - Synesise new stand 3, Extension
Pe SEBEREEBEN
Thermal Cycler
www.bankofbiology.com
PROCESSES OF RECOMBINAN A TECHNOLOG
LMinsertionfofjRecombinant/DNA\intojiost{cell)oreanism aan
* Using any methods, the ligated DNA is introduced into recipient (host) cells. They
take up DNA from its surrounding. bankofbiology.com
+ Ifa recombinant DNA bearing ampicillin resistant gene is transferred into E. coli cells,
the host cells become ampicillin-resistant cells.
+ If the transformed cells are spread on agar plates containing ampicillin, only
transformants will grow. Untransformed recipient cells will die.
Ye
E. coli Toners cell
phernid (DNA) Host (recipient) cell
© —
$ Nutrient medium
(O ©) Amplcillin Ampicillin-resistant colonies
Transformed cell Non ED cell
www.bankofbiology.com
LMinsertionfofjRecombinant/DNA\intojiost{cell)oreanism aan
* Using any methods, the ligated DNA is introduced into recipient (host) cells. They
take up DNA from its surrounding. bankofbiology.com
+ Ifa recombinant DNA bearing ampicillin resistant gene is transferred into E. coli cells,
the host cells become ampicillin-resistant cells.
+ If the transformed cells are spread on agar plates containing ampicillin, only
transformants will grow. Untransformed recipient cells will die.
Ye
E. coli Toners cell
phernid (DNA) Host (recipient) cell
© —
$ Nutrient medium
(O ©) Amplcillin Ampicillin-resistant colonies
Transformed cell Non ED cell
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
3 Obtaining the Foraizn Gana Podue |
+ The ultimate aim of recombinant DNA technology is to produce a desirable
protein.
+ Ifa protein encoding foreign gene is expressed in a heterologous host, it is called
a recombinant protein.
= pat,
2 Q Y
DAA > == = c
Construction of xpresion plaid
LE) rexprewion Recombinant protein
Prokaryeticexpresion
www.bankofbiology.com
3 Obtaining the Foraizn Gana Podue |
+ The ultimate aim of recombinant DNA technology is to produce a desirable
protein.
+ Ifa protein encoding foreign gene is expressed in a heterologous host, it is called
a recombinant protein.
= pat,
2 Q Y
DAA > == = c
Construction of xpresion plaid
LE) rexprewion Recombinant protein
Prokaryeticexpresion
www.bankofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
3, Oxtefining tha Farga Gana Palas )
+ Cells with foreign genes can be grown in laboratory. The cultures are used to extract
desired protein and purify it by using separation techniques.
* The cells can also be multiplied in a continuous culture system. Here, the used
medium is drained out from one side while fresh medium is added from the other. It
maintains the cells more physiologically active and so produces a larger biomass. It
yields more desired protein.
Fos OS |
ANS &
1kofbiology.com
3, Oxtefining tha Farga Gana Palas )
+ Cells with foreign genes can be grown in laboratory. The cultures are used to extract
desired protein and purify it by using separation techniques.
* The cells can also be multiplied in a continuous culture system. Here, the used
medium is drained out from one side while fresh medium is added from the other. It
maintains the cells more physiologically active and so produces a larger biomass. It
yields more desired protein.
Fos OS |
ANS &
1kofbiology.com
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
SAOPtainingithe Gene} )
Bioreactors bankofbiology.com
* These are the vessels in which raw materials
are biologically converted into specific
products, enzymes etc. using microbial, plant,
animal or human cells.
+ Bioreactors are used to produce large
quantities of products. They can process 100-
1000 litres of culture.
» Abioreactor provides optimal growth
conditions (temperature, pH, substrate, salts,
vitamins, oxygen) to get desired product.
www.bankofbiology.com
SAOPtainingithe Gene} )
Bioreactors bankofbiology.com
* These are the vessels in which raw materials
are biologically converted into specific
products, enzymes etc. using microbial, plant,
animal or human cells.
+ Bioreactors are used to produce large
quantities of products. They can process 100-
1000 litres of culture.
» Abioreactor provides optimal growth
conditions (temperature, pH, substrate, salts,
vitamins, oxygen) to get desired product.
www.bankofbiology.com
PROCESSES OF RECOMBINAN A TECHNOLOG
5JobtainingithelkoreieniGenelbroduct, |
Bioreactors
* Most commonly used bioreactors
( > are of stirring type (stirred-tank
Acid/Base Motor reactor).
for pit Foam
control bbraker + It is usually cylindrical or with a
Steanvfor Fiat bladed on
steriisation eee. curved base to facilitate the
‘on mixing of the reactor contents.
L een) The stirrer facilitates even mixing
and oxygen availability.
Alternatively air can be bubbled
through the reactor.
a) Simple stirred-tank bioreactor b) Sparged stirred-tank
bioreactor through which sterile air bubbles are sparged
www.bankofbiology.com
5JobtainingithelkoreieniGenelbroduct, |
Bioreactors
* Most commonly used bioreactors
( > are of stirring type (stirred-tank
Acid/Base Motor reactor).
for pit Foam
control bbraker + It is usually cylindrical or with a
Steanvfor Fiat bladed on
steriisation eee. curved base to facilitate the
‘on mixing of the reactor contents.
L een) The stirrer facilitates even mixing
and oxygen availability.
Alternatively air can be bubbled
through the reactor.
a) Simple stirred-tank bioreactor b) Sparged stirred-tank
bioreactor through which sterile air bubbles are sparged
www.bankofbiology.com
PROCESSES OF RECOMBINAN A TECHNOLOG
5 Ole tha Foreign Gana Precust
Bioreactors
2 = Parts of a Bioreactor
+ An agitator system
Acid /Base male 5
am = + An oxygen delivery system
Ela tte + A foam control system
rol
+ A temperature control system
L siente ur |
+ pH control system
a) Simple stirred-tank bioreactor b) Sparged stirred-tank .
Sampling ports (for periodic
bioreactor through which sterile air bubbles are sparged
withdrawal of the culture)
www.bankofbiology.com
5 Ole tha Foreign Gana Precust
Bioreactors
2 = Parts of a Bioreactor
+ An agitator system
Acid /Base male 5
am = + An oxygen delivery system
Ela tte + A foam control system
rol
+ A temperature control system
L siente ur |
+ pH control system
a) Simple stirred-tank bioreactor b) Sparged stirred-tank .
Sampling ports (for periodic
bioreactor through which sterile air bubbles are sparged
withdrawal of the culture)
www.bankofbiology.com
PROCESSES OF RECOMBINAN A TECHNOLOG
& Downstream Procassihyy
“upstream processing” | “downstream processing”
Pressure, pit °C, PLE, * It is a series of processes
such as separation and
purification of products after
the biosynthetic stage.
* The product is formulated
with suitable preservatives.
Such formulation undergoes
clinical trials and strict
quality control testing.
www.bankofbiology.com
& Downstream Procassihyy
“upstream processing” | “downstream processing”
Pressure, pit °C, PLE, * It is a series of processes
such as separation and
purification of products after
the biosynthetic stage.
* The product is formulated
with suitable preservatives.
Such formulation undergoes
clinical trials and strict
quality control testing.
www.bankofbiology.com
paxmat
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CNACUÉO Lui tls aan da nk je E misa in PS 202 =
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Del ie kl p
is “N [alıN Malin ada
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© tava leh Shanyavadagau m
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=m4=44 Mlyabonga Mia lsd
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CNACUÉO Lui tls aan da nk je E misa in PS 202 =
Hina =, 3 PES mar
a ‘=e. A
Del ie kl p
is “N [alıN Malin ada
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