Uploaded by Dorothy Ma Sieu Yieng

Genetic Engineering: Human Insulin Production in E. coli

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Genetic
①
Isolate
engineering
(of
human
gene
from
6
cut
(b)
made
*
DNA
large
in
cells
.
number
identify the location of
to
already
processed
to
make
>
,
SS
Use
=>
DNA
>
restriction
>
into
computer
DNA
>
0
.
05
contains
:
of
to
the
produce
CDNA
human
insulin
synthesiser
machine
fragments
are
the
with
with
later
gene
make
gene
joined
polymerase
thermocycler
gene
Cusing
(PCR)
gene
DNA
am3
of
The
copies
reaction
H
template
.
many
-
~
.
fragments.
chain
as
DNA
sea
Make
②
mRNA
-
nucleotide
together
the
uses
key
in
cDNA
polymerase
enzyme
ends'
synthesise
-
ds
DNA
.
sticky
(a)
.
DNA
transcriptase
make
to
exons
DNA CcDNA)
complementary
transcriptase
reverse
the
containing only the
reverse
*
& is
.
chromosome
mRNA
the
found
are
the
is
B
of
mRNA
SS
.
cytoplasm
trying
than
mRNA
use
=>
from
they
on
gene
.
.
mRNA
easier
insulin
human
produce
MRNA
because
*
.
chromosomal
from
isolate
-
insulin
to
E coli
polymerase
pH buffer
free
2
deoxynucleotide triphosphate
of
types
strand
,
one
DNA
for
primers
(one
downward
(dNTPs)
for
strand
upward
.
Stage)
°
95
At
At
primers
SSDNA
by
3
the
7202
DNA
,
free
dNTPs
heat
stable
.
so
>
so
-
Insert
does
not
need
to
into
vector
open
-
Cut
the
gene
plasmid
to
step
-
polymerase
during
(such
as
is
,
efficiency
.
maximum
for
replace
the
with
birds
and
cDNA
the
bonds
H
to
specific
either
produces
.
cycle
each
plasmids
,
viruses
,
same
obtain
restriction
the
plasmid.
enzyna
'target
of
seg
with
4-6
ends'
sticky
site/restriction
base
or
by
simply
reform
.
ends
ends'
of
cDNA
with
cDNA
with
the
sugar-phosphate backbone
plasmid/recombinant DNA
to
form
ligase
pairs (bp)
'blunt
.
plasmid
Sticky
site'
complementary sticky ends of
Some
plasmids
.
complementary base pairing
plasmid
DNA
strand
new
produce complementary sticky ends·
enzyme/restriction endonuclease
identify
form
the
1
restriction
Mix
of
tag
as
centrifuged the bacteria to
&
containing
-
pairing
.
.
cut
-
synthesises
such
optimum
-
-
,
high
the
from
used
has
liposomes
one
.
polymerase
>
base
.
with
bonds
hydrogen
complementary
polymerase
DNA
-
form
DNA
s
Extension
:
using
to
denatured
is
DNA
Stage
*
DNA
60 %,
of
*
ds
,
Annealing
2
and
At
C
:
Stage
3
Denaturation
:
joins
the
.
plasmid by their
,
recombinant
cDNA
*
①
plasmid
>
Mix
>
cools
#
About
before
of
1 %
by
its
bacteria
.
coli
shock
the
has
bacteria
.
Cartions
high
in
heat
taken
be
to
E
into
bacteria
and
mixture
the
plasmids
-
plasmid
with
.
marker
genetic
the
recombinant
Insert
-
and
promoter
own
plasmid together
into
inserted
be
should
the
to
solution .
chance
of
cell.
taken
the
up
recombinant
.
plasmid
transformed
Identifying
5
-
GUS
gene
conversion
of
B-glucoronidase
:
colorless
of
non-fluorescent
So
transformed
fluorescent
P
gene
so
coding
placed
6
Transformed
to
when
,
for
transformed
to
using
genetic
P
bacteria
would
fluoresces
bacteria
colored
fluorescent
green
UV
,
P/
S
.
produce
under
conversion
colourlessS/
the
to
E
the
.
produced
with
or
.
P
fluorescent
fed
markers
(GUS) catalyses
coloured
world
bacteria
non-fluorescent
-
bacteria
light
are
green
protein (GFP)
when
.
grown
in
large
scale
.
fermenters
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