IstepVII selectionofasuitabl lvectorf

Vector will be the term used for a

ca who will help in delivering

the
desire ie the gene which
will code for the desired product
into a hostel where product
formation will take place
6

example of a vector can be a plasmid

molecule which will help deliver
to
a desired
genre in a bacteria cell
where desised will be
the a product formed
characteristics ofmy a suitable vector
b

SEE The vector molecule

should have small
size because the
hosts not accept
may
large sized vectors

orisit ie
origin of replication
site which would
 ensure that the vector
can make its own

copies along with the

copies of the desired
gene attached to it
independent of the
host's chromosome
osi site must initiate replication at

regular intervals so that the vector

able to
will be make
high number of
copies of itself as well as the
desired gene in cell
the host
The presence of ansop gene replication
of plasmid gene is also essential for
regulation of replication pass
presence of
selectable
markes selectable markers
mean some special
features of the vectors
ex antibiotic resistant
genes of plasmid vectors
which will help to identify
whether the host cells
 have accepted the
vector or not

Multiple cloning site

M nte
this refers
to
parts of the
vector where recognition
sites of many Restriction
endonucleases are present
At these parts of the
vector a lot of desired
gene fragments obtained
with the help of a
variety of R E can be
attached and once the
attachment is done the
vector will the
carry is
desired
gene fragment
the host cell
 Diagrammatic representation of
chaste
Tetracycline
Resistance gene Mcs Poly
sequence linker
tetro Bam
or

Ah
t ai P iii It
Erection
cognition
co Rts vector PBR3227
chat 2 A plasmid gates of
Hind't many
Itt
restriction
9 ALI wipes
Ori replication like salt Bam
of plasmid Amp
site i.e
gene
withrecognition
Ampicillin Resistance
siteofputt gene sequence
with serognition sequences
sestrictionerm E Punt
of Pst enzymes
Mcsas Poly linker
Examples of some vectors used
tmoden chnologyt
i
plastids these are circular
autonomously replicating
dsDNA molecules which
are used as vectors to
deliver desired genes
in bacteria hosts
 Names of plasmids
used as vectors are

P
PERI
plasmid stthesia plasmid prepared
by Bolivar in University of

Rodriguez 322nd California

plasmid synthesized in Lab no 18

by them t
a Its selectable markers
its selectable are am.PL gene and
markers are
E gene
amp gene and and its size is
teth gene
and its size is
much smaller Kbp
approx If Kbp
kilo base pairs
OF
gene
i Bacteriophage viruses

the viruses can also be used as vectors

to deliver desised genes into bacteria
hosts
The commonly used viruses for this
purpose are d phage virus and

M I3 virus

desired is attached to the viral

gene
and the viruses ate liver
genome
their genome along with the desised
gene into host bacteria

genome has sites

a phage cos or

sticky ends or cohesive ends and

it can also create complimentary
sticky ends on the host chromosome
making integration of its genome with
the host chromosome nose
efficient
iii
cosmide synthetic flab psepased
A
plasmid on which cos sites of X phage
are also attached so it gets
incorporated into host bacteria's
chromosomes easily
 v BAI B al artificial
chromosome
synthetic chromosome with essential
genes afor survival of the bacteria
and Large sized desised gene
It is inserted in a bacteria host
cell whose native self chromosome
has been taken out first
BAI is a suitable vector it the
size of desired gene to be inserted
into bacteria host cell is Lege

C Retrovirus vectors vectors

used as

to deliver desired
gene sequence
a

into an animal host cell

Retroviruses have high capability to
infect animal cells hence
theyinto
ensure
delivery of desised gene
animal host cells
however
effectively
Retroviruses must te non
disease causing to be considered as
vectors ex Reo virus Riya E sia
virus
orphyan vines ie a obtained from
 human gut end on
having RNA
genome
ie.se isns not
known to cause disease orphan
any

D T

Agrobacterium tumifaciens beaten a

is also known as natural genetic

engines plants
of because it
has a
plasmid in it called the
Tumor Inducing plasmid
t
plasmid contains TDNA_sequences
or
transfer DNA sequences which
help the plasmid A Fecht
of dicot plant cells
genome
and plasmid also has r virulence
region DNA which
or a
sequence
can induce
tumors in the infected
plant cells which are soft cells
1
So the bacteria causes crony golf
disease root tumors in infected plants
 i

plant
cell chromosome

A plasmid is obtained from a

bacteria cell and the plasmid is

ie vi
first disarmed its region
is semoned tht T DNA sequences
are left intact
L
and then a desised gene
which needs to be delivered into
plant all for its expression
is attached in place of vise
segion L
the desised
gene now
gets easily
plant into cells
withchromos me
integratedthe help of TDNI
sequences of the plasmid
1
So expression of the desised gene
occurs and the protein products
can te obtained from plant Prost and
the plant host does not suffer
from any tumors as tree plasmid
vector was in disarmed form
D KAIT yeast n cial chneosome
It is a
syntheticflab
poepased chromosome having
centromere and telomere sequences
of yeast and desised gene which
can be
upto temp million 2
base pairs long is attached between
these centromere and telomere
sequences
Now the artificial chromosome
can be introduced into yeast cell
where it is
easily accepted and
the large sized desised glue is
expressed proteins are
formed
 YAI is the professed vector
if the desired genre is a
huge
eukaryotic gene x a human gene

M
hydesised gene
Eqq3zfg2g upto smbD forms
centromere protein
products
a toast

µ
3kg3
D
from qed
felonies
YA c vector

for
the gene protein
expression in cell
formation host
yeast

viii LYEIT ie
jeers episomal phased
plasmid with ori sites ie
one site which helps the plasmid
to replicate in prokaryotic host cell
and another osi site which helps
the plasmid to replicate in
eukaryotic
host cell
so such plasmid rector can te
used to deliver desised gene in
different types of host cells
k
such vectors are also called
shuttle vectors

is
Even
YAI
an
example of
shuttle vectors
In modern biotechnology the
most comm
only used vectors
are
plasmids and the most

commonly used hosts for expression

of desised genes attached to the
plasmid vectors are bacteria
1541
gene fragment with the
fadditionofthedesised
selected vector Plasmid

A few plasmid molecules are taken

in a test tube plasmids can be either

p or
puc 18
II R E is now
type
added into this test

tube
Allplasmid molecules
get at open at the
recognition site of the particular
type I R E used 4
Now the desired gene
fragments are alsoput
into this test tube
along with DMI ligase
enzyme
I
2
types of plasmids will
be obtained in this test
 tube is

a Recombinant
plasmids plasmids
do I

I tfu
iiÉIÉI E
THIFFHH amprgee Etttesamprgere
tenant I enzyme
Banana
added in added in
thetuse the tube
the the
plaswittttasampRgene plasmidtttafsampfere
opened up opened up

desired gene
gene
havingcompli Ides'ised
having
compl mentary
overhangs is
mentary is
overhangs

Litton
test tube along
ligase
Lithonia
test tube along
ligase
But the open ends of The desired
the plasmid close gene gets
again without receiving incorporated
the desired gene at the site
t where the
so the plasmid is
cut was
said to be
E ligated introduced
1 by
Barn HI
the d designed
teth
at ampgene
EtEI9m
EETEEI
g.EE
ThetampRgene
plasmid is in said
to te se combinant

probability of formation of both

seligated recombinant plasmids
is
equal
The RE chosen to cut open the
typeof
plasmid must be the same which was
used to obtain the desired fragment gene
which is to be added 6 the plasmid
otherwise the single stranded
overhangs sticky ends of the
open plasmid the desised gene
fragment will not be complimentary

Religated plasmids will have both

selectable makes in active functional
form because the selectable maker
which was cut open by the R will
return to its functional state due to
seligation
However the recombinant plasmids
will have one selectable marker in
functional form the one in which R E
did not introduce any cutbut
the other selectable master is
in
innally inactivated from
ie will be
permanently inactivated
decent insertion of desised gene
fragment at the site where the
cut was itroduced by R E
 If the plasmid vector chosen was
PBR 322 plasmid then the seligated
plasmids will be having both ER gene
and out
gene
in
functional forms
while the recombinant plasmids will
be
having fin functional
form teth gene is insertionally
the above
inactivated form in example
where Bam Ha is the R E being used
If the plasmid vector chosen was
put plasmid then the seligated
plasmids will have both had gene and
omf gene in functional forms as
RE opens up the hay gene sequence
which closes back on
relegation
while the recombinant plasmids
will have amp gene in functional form
and lacz
gene in insestionally
inactivated form as desired gene
sequence will be inserted in it

So at the end of step VI 2types

of plasmids will be available in the
test tube seated recombinant
 and their selectable markers are
their
differentiating features

Relegated
plasmid
87 Recombinant
plasmids

fsteputt Introduction of the plasmids

into host cells ie bacteria
in which the desired
will be expressed and
gene
products will be formed
t
It impossible to separate the
is
seligated plasmids the recombinant
plasmids which are available at the
end of step It as their sizes are only

minutely
different
hence both types of plasmids have to
be inused step VII
methods of introduction of plasmid
 Vectors into host bacteria are

Egan Ibiolistin

c t q I
Gene gun
the
introduced Into
host cell attached to a
gold
or
tungsten particle both of
which are inert heavy metals
providing enough momentum to the
DNA so that it can penetrate the
cell wall of host bacteria and
they do not interfere with the
host's metabolism as they are
inert

Dnamicroinjection

pipette EEEamia
L micro
holds the syringe 4
host cell micro needle apparatus to
firmly in position inject the Dart A in the
host cell
 Transformation All plasmids
obtained at the
poured end of step
nfontents a plate over as

poured
Lingi
pwning colonies
V the
with over
culture plate
muggy
colonies
I
bacteria or not take up
the
may may
plasmids
The bacteria the culture
growing on

plate may be given various types of

treatments to enhance their chances
of up the plasmids
taking
the traced bacteria are also called
competent bacteria
2
and the treatments involve
i
Heatshock exposing the
ie
bacteria to 421 and
alternately
of temp for a
few seconds
repeatedly
 ly
Je
this creates poses in the bacteria's
cell walls
easier
making entry of plasmids
ii Ectroporation exposing the bacteria
to mild electrical currents which creates
minute poses in their cell wall
making
entry of plasmids easier

in
Adding ca ions and or P EG Poly
the
ethyleglyofalong In
plasmid solution on the culture plate
having the bacteria
host helps
in enhanced chances of plasmids
sticking to cell walls of hosts with
the help of cod PEG so the
of host the
probability accepting
plasmids Lis higher
this card PEG mediated uptake of
plasmids is also called transfection

Transformation is the most commonly

used procedure to introduce plasmids
 into hosts
At the end transformation
of
process types of bacteria colonies
will be present on the culture plate
ie

Non trans formant Trans formant

bacteria colonies bacteria colonies
I 2
bacteria colonies Bacteria colonies
which do not which accept take up
the
accept any type any of
2

of plasmid ie types of plasmids

any
egg my
nor recombinant s
And these colonies
can be further
divided into styles
2

I
Non recombinant Recomb nant
Trans formant transfer

fyyy
mants
bacteria colonies to
Bacteria
which accept colonies
which accept
Religated
gym mi.de
o The bacterial colonies of all three
types non transformants Non
recombinant transformants and
Recombinant transformants will
have distinct features and can be

easily identified distinguished

If the plasmid vector used was
then
PBRIZ
Non transformanthon Recomt
Recombinant
bacteria colonies binant
which do not accept transformant Transfer
want
colonies
anytype of ABRI colonies
which have
will te sensitive
taken which
to both ampicillin up take up
and tetracycline Religated the
PBR 322 recombinant
antibiotics
will be PBR 322
will te
resistant to
resistant
both ampicillin
f to
ampicillin
tetragline but will

Kauai
sensitive
to
offtheplasmidearnedwas
puc is then

Non transfer Non Recombinant Recombinant

want colonies transforment transformant
will be colonies will colonies
sensitive to be resistant will be
ampicillin resistant to
afflict
to
and will have ampicillin
and will also a
functional but will have
not have lez gene non functional
Lac 2
gene in them iTsertionally
Them inactivated

these