[VII) Selection Transformant

of. from non transformant ;

recombinant
separation of, transformant from non-recombinant
transformant.

NT :

Non-transformant
NRT
=

Non-Recombinant transformant
RT =

Recombinant transformant

Insertional Inactivation : Inactivation of selectable marker gene

due to insertion of foreign DNA.

↓
M-RNA
~
2 Protein In need see

↓ I

Phenotypic Expression . No Phenotypic expression

(a) Using two antibiotic resistance genes =>

pBR322 .

Non-Recombinant Bartl
Recombinant Plasmid
Plasmid gene of
tetR BamHI ampr interest
ampR ampe S
.

fetR tetS

·
o ·- NT

·00
·> -
NRT

he · -> RT

O ⑨
NT lack ampR gene ,
hence, Ampicilia will be added
eliminated .
get
S

·
O
O
O Parent Plate .

⑨ [ o

?
o
·
(

Replica plating on culture

medium containing tetracycle
RT's lack tetR due Y

to insertional inactivation
T
eliminated in
&
get O
retracycline containing [ in
medium ⑨
·
.

from parent plate.

RT's isolated and used for further
are
process .

Drawback
-

It is a cumbersome process because it is difficult

to attain accuracy a prevent contamination during
replica plating.
-
Also , maintaining two cultures is time
consuming.
(d) Selection by one lack gene and 1 antibiotic resistance
gene
-

pUC8 .

Lactose (chromogenic
ri ~ lac
z
gene
Subtrate) .

-
B-galactosidase
Glucose + Galactose
(x gal) = blue
NR-plasmid
-

.
 Recombinant Plasmid-Produce Transparent/
white colonies

-- > NT
· - RT NT get eliminated
-
NRT X-amps
... &
- ·
& :
&
incubated in E
-
- -
> · Y
G -
M
-
ampicilin w
&
-
G &
-
chromogenic lactose & ·
- - &
medium
i
/ &
& E
NRT cells
W
1 RT-
blue
appear form while
colonied due to
insertional inactivation
of functional lat 2
gene
B-Galactosidase >
-
called Reporter Enzyme .