BIOTECHNOLOGY PRINCIPLES AND PROCESSES

LECTURE 07

Examples of Vectors
Plasmids Retrovirus Agrobacteriumtumefaciens etc

Plasmids are extra chromosomal DNA

double stranded Cds

self replicating
Chromosomal
DNA t Plasmid

Bacteria

DNA Ligases helps in with

joining foreign
o
gene
vector

Vector PBR 322

plasmid BR 322
p
specific no for a
Boliver Rodriguez particular plasmid
 Eco RI Hind
BamH I
PUNI
amp
Pst I te't
salt
p BR 322

guido tfEpo IReplication

proteins
of
II
Construction Identification of Recombinant Transformant

Bam n bg gene of
big we
a
Interest
try to cut the Plasmid
ampR tear
to create Recombinant II Recombinant

DNA by joining gene

of interest
Be
ampr tet

I Non Recombinant
 The next step will
transforming HOST
be 3

possibilities are there

E coli
I
NON TRANSFORM ANT
d d

Egg
ie Non recombinant DNA has been transferred

E coli
E
TRANSFORMANT RECOMBINANT

i e R DNA has been to host

transferred

Note
Recombinant Non Recombinant is used for Vector
Transformant Non Transformant is used for Host
Culture of the E coli in AMPICILLIN Medium will lead
to death of Non trans formant E coli because
of
lack of any plasmid
Ecolic I

É
33
Eocoli TI

Culture Ampicillin

create a Replicaof 1st culture plate we place

with tetracycline
REM

2
Culture t tetracycline
 In the 2nd culture
plate with tetracyline Only
o

the non recombinant survive because they have

intact Tetracycline which provide them

gene
resistance whereas RECOMBINANT die because
due to the insertion of foreign gene in Tet gene

the
genegot damaged

We can still locate the position

of Recombinants
o

from culture I take it out

grow separately

o All in all In Ampicillin medium selection between

transformant Non transformant occur

In tetracycline selection betweenrecombinant

and Non recombinant occurs

o E coli does not resistance

carry against
antibiotics like ampicillin chloramphenicol
tetracycline or Kanamycin etc
Note Alien DNA is carried out at a restriction
site present in one
of the two antibiotic

resistance
genes