12.

00

13.00
Clonmg Vectora;

Thy ore cariers or vehicles o desired NA fragmentai

14.09

DNA).which Cm umdeigo 'mdependent replicaiôn

ant copieks
A bactiium
desived qenaq paamdabacensog
1599
hich com yeplicata m tuo elilerent
erent bosts
16 called ahutte vector.
Plasmida Gd
Cnd batexiohages have the to replieale
within baeeial cella indeependent
pendent ethe cotol o
nO%omal
hromosomal NA
 12.0

Thexefoe., i we
we ore. abe recombinea piece cy
to
a DNA w

bacteraphage genome aY plasmid DNA, we can mwbiply iUS

numbe
*VectoYs
thal thy
coual to the cop
ised present
help
are
number
al
easy unking
, o
engineexed
foeign
o theplaSmid
in
NA Ond
Auch awa
selecbon
ooY

a
bacte

wd
14.00

xecombinah fom non- Yecombinanb 15.00

laFeatuyea Required
Required în
in Üoming Vectora;
A cdoning vector must have ia own orign exeplicationor)
electable narkex Cmd
preleraaly a cmgvecegnibim aiir 17.00
fsr Jonin
Types Cioning Vectors;
iClonimgvectors are four peo plasmida, viruses cosmi
Sund arificialchDmOSome
-PLASMiDS -
PlasmidA Qremal
smal extracellular CKCular DNAs which
cOaY exhachromasomal genes in bacteria omd some -fungi.
Tyreplicali imdependently
kTh beot known plasmid
plasmid vectorgwhich
vectorewohich are aloo Cvailae
ore pBR
MTcommerAally wTFS 322 Sond.
AUGUST
TF SS
W M I M W S S M TO W
TFSSM T W
12 3 5 674 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
 WEDNESDAY 22005

them have
Both
DRDOP sCheichiacoli.
been modified from naluial plaimdo e
09,00
They have the follwmg feature.

10.00
i) Origin ef Replucotion (Ori)
This s the seauence from whee repication alarts nd ny
piece o DNA when nked to thii sequene vetpensible fe

heototing Cam be
can. made to replicali ithm the host cel.

The seuenie is alao reeponible for controlling the copy

Nhumbl ot the linked NA.
TheefoYe, the selected plasmid vecor should have am
12.00

"ongin e eplication that supports high copy umbex.

n0Alcctable Morker;
Its a
Yepater gehe that helpa in idenhfmg Cmd ediminatmg
14.00

non ransfoYmants ondselecively penmhn the

the tramsfomamta. (Arificial leton).
the gene encod mg for ontibiaic reei stamce&uch

ampiiun, Chloxamphenica, ttaym e considerc

Usejd ao slectable martku fos E co

ts The nomal E. coli do not [Link] any
mt
Te these omtibiohcd.
eg. pRR 322 has tuD Tesiabante qenes, ampclin
18.00

etragclin xesistonce gene while haa onky ampicllin, pC-i8

delectable marke is ment for distig uishmg a ccombingnt
NA. Thus the
from non-Yecombinant elimihating
helh
nom-tranfomed back ial cela feom tansfm med badnal cella

t Cloning
9n order o
Sitea
nk the
freigh
DNA the ve cfor needs to
Aalhae veay
commonly
fe pre ferably single, gecognition Aa fr
fr
ULY he M ASedYe
MT WT shicton
M.T enzyme SSM
23 4 5 67 8 9
S

10 11 12 13 14
FS
15 l6
S

7 18 19 20 21.
F

22 23 24 25 26
WT
27 28 29
S

30 31
 JULY

2005 THURSDAY 21|

moYe thm me
ecognition sia will euse
DA generall DNA
several fgmente amd wimake
gene cloning complical. A
* In pBR 32 alien DNA ugalid
fe generally n tne
G
ea o Aile o BamHI resis
tetracycdm resis tonce gene 10.00

>* Thus Yecombinnt plasmiddont posteszes tetracyclin

Yesistance but Connue to ba ve. ampiciin yesictance
* nacbvaton qene due to msetion alien DNE
18 called Tnseinalinactotien. NOON

for Plamts cmd animala

Cionimg VectorA 12.00

Tumor inducing plasmid Ti plasmid orIDNAJ aoil

bacteium Aqrobacterium tumifadcna has been made non 13.00

virulent by modifng the tumouY inducm gene T DNA

insetod in T- DNA Yegion s the
14.00

C
to make t rnative T
plasmid
Yecombinedo vectox 18 nQw allowed sol500
cloring
cella tissue clture.
the ploot ox n

The plasmid with the desired inaeted passeg ralto

ne
varioua parts expre sses te ect.
Aqrobaclerium tumifacie08 infecta
non oLUent
only dicat plonta
plaonid com alA Cause V
BE
+he oneA wrth modaecd i

genetic ichangta
monocoa. 18.00

k Retrovirukes which have the to tmsfom morml

animal ella inbo CancrOUA celld (e Simin viru y) cm
be for simias purpose im animaa.
wsed
eDisaYmed rehovix uaeghave also beenused to deiver
desivable gens imto onimal cello
a hNA fragment hau been Ligalkd into
a suitable vectoY it i8 transfered into bachenal, plemt o
hogtwhee
onimalhogt miplles.e AUGUST
M M T W TF SS t
M TW TF SS M TW W
3 10 1 12 13 14 15 16 17 18 19 20 211 22 23
28
5
24 25 26
2
27 29 30 31
 JULY

FRIDAY 2005
Cosmid
08.00

They are plasmid im whioh phaqe lambda cOB alas have.

beeninseared
09.00

A cosmid can packecd in the phage coat

uselul in fiogmeat.
10.00

Competent Host -
11.00

*
NOON
Competence
Teom outside
s the
abiliy bo pick up genea cndplasmids

(e ost specie of bacerîa, inchuding Ecoli, take up anly

nnited amount DNA Inder nomal eiYcumAtamced.
te Sn odea to transfom these species icientdy,the baclena
have to
bo mdergo some physical and ar chemica taeatment
that enhances theis ahillty to take uþ NA
14.00,
k DNAis a aydrohylic
a motul. hene caot pat through
cell membrane.
Mge Compence Cm be enhaned
treating
he hot cell

Loth a
specijie concentabon dalent cabin like caleium.
A solution b 5O mM calcum choride is wsed, althsugh
othu ubidium lA,re aleo notably chlonde, ective.
Wk 9t simuat attchment et NA
add over theAeceptoY RihA of thwceu wll
1800The actual movement. o NA fnto conpeent eell 18
tempezature to 42°c,
Aimulalzd bybriaikng the

Othe MethoJa:
4The desjred gene aswel Ye cOTmbmant NA can bei passed
Intoplomo,animala buman

arnimaloceA
r
meana
cels througk
micropipetho imts ucleua ar
5 Gene gun

1 2 3
MT
45 6
w

7
F

8 9
Biousti

O 11 12 13
inl Par hcle
14 15
S
1617
S M
18 19
nwhere
2021
TWT
TF SMtungeston
22
S

23 24
F S S

25 26 27 28 29 30 31|
 JULY

2005

geld partides caatzd wth desired qenea are

SATURDAY

bombardec
28
08.00

into plmt
celo with greatforce.
c)Disarmed pathoen. com be used as vectoYA for trmsfe
Tecombnmt NA
into the hast.
09.08

10.00

Proce ss es Of Recombinont NA Technelog l.00

Recombinant DNA Technoloy. invelved Aeveral alzpa QA folloo

NOON

Sep I) Tsolaion o the Genetc Matzial (ONA)

12.00

bCulhure cells, from which the desired genes cx DNA

DNA egment 13.00
aYe to beobtaned, Qre sepsJecd.
The cella are hrealzdoth
trealed ortt yso zyme (fox baczyia),
sozyme baczria), cellatane (f
kThe trealedcella
(
omd pectirase (forplont cells
ore. homogenised and centifugcd
)
nd chiinase (foy fungal celas C
T
Yuptue the cell aswet as nucleaa envelope.," 15.00

The homaqenised then trealzd Loith

product is poteases,
Yibonucdeaseg laseA ond ibases
16.00

amylase
Othe molecuUe an be Yemoved treatmente
appopnala
and purifed DNA bimaay preipitale the 17.00

chled ethan
sk DNA appeaA as mass svey fine thseada. and ca
18.00

Pasale spooling OYlwimdm oYe a

ineappliance

Cutting at dpecii
of ANA
LocatimA;
Restichion
enymeidigestionaare perfoxmed imubating
pusified DNA rmolecwest oith the Yshicin nzyme
opimalcondiins nc enzme.
hareeaeelechrepharesis
W used toeheek
SS the prayGn
123 45
M
89
W F M W TF
7 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
 JULY

25 MONDAY 2005

enzme
8oheyestictima
ne cdesired genes are
diqestion.
identiyed
identiyed and separaläd
The genetic maleial meat for trmsfe to notha
hich iA
Bganism callcd passnge DNA.
The process wth he vectoy NA alool vehice
10.00
pocessiA Yepealzd
DNA)
1L.00

SrepI) Formation e Recemb nmt or rDNA

NOONe oth the Passange cmd vehicle DNAS ane treaad

alad separaay
Leith the same relticbiom endonuclease to podules complem
itay sicky cnd&
out gene o mlrest the soumce
foom tbe
forom NA God

re ond jomed together wth the

helt r
vec boY
DNAligase Ty
mixed
ligase, molecular
ehecked by use s alkatine phoshala.
g)
*The union o plasmid r vehicle DNA d the desiyed
15.00

pacsmge NA fragment prsducaa

Chimera
16.00

17.00
Aep ) Amplificabin Genee
lateaest

Ot posible to gene e intesest to approxioalg

18004 bintimes within a
apon aine day thaough palgmeca
ehan eacbon ( kany Mullisy 1985).
with
DNA Can be used to ligala
Šgment
the vecbor fa fomatiGn e DNA nd Ua
elaning

Insrtion b RecombinanttiANA into ttodtas

k Both oehodaaCe khon bo
direct Gnd imdirect
celhdtou
ntrducehe deciYed gene into Bast
glYSn
5MT aommonmethoc,
WT S the desiYed
M TWTFS MT TFsgene is
F
MT gala
TFSS31
S
S w S
10 17 24 9 11 12 13 14 15 l6 18 19 20 21 22 23 25 26 27 28 29 30
2005
TUESDAY 26
a plasmid vector. 08.00

bacenatcells Ecoli) are made coopelent.

The plomidree gene
DNA nd ampicillinyesistnt
The plasmid havmg

r
qre nterodueed 9 theiY meciue.ost3owb G
bacenal cells, the bost
k As thee plasmd coter the 10.00

become ampicin sistont

cell
fosuse m breacor.
aYe now Aelecled
-k The trmsfmed cella recombinamt,
tance gene is used n delecti
Yess
calledselectable marke NOON
wt i&
E
Gene Product,
Aap )Oblainñng thePoyeign
aim recombinaat bistehnsloqy ia D prsdHe
e
* The major
to hur
a desired° biochenical ooproteinuseful
Dbinant SNA echntlo
As t iahpoduced through Aeconbinant
cetbscthe ig called Yciombjnmb pduct
mside heeralsgous
a
host
ochemjcal.
bi
C
14.00

c
proteen
cells cloned gened mleret may be T
harbourng
Acale n the labreatoy
Poon eithe
batch
laxge s cale in bioreactors [either through 16.00

Culture o coNtinuoud culture systemL

be the desired
The culures used foY extracting
for extractbing

protein nd then pundy mg it usg cifeaent separaom

techniques. 18.00

Aip )Aoonstreamig Processhgi:

imvolve puifahirr
Dawnsreaming procesaing piocesm separation,
Gnd presevation pradicts.
cellalar mass of imoculum, the rest is
*Afler leavng a part
crushed andchemically treazd to separa the product

laThe separalkd pcdut s purifed amd then fxmulalad oith

Auitable presevahvek
Cimical brotls are carred oLut to know ta we md HUsT
MT WIF SS MT WT SS W IFS SS
J2 3 4 5 67 8 10 11
F

12 13 14
M

15
T

16 17 18 19 20 21
S M

22 23 24
W

25
F

26 27 23
M

29 30 31
 JULY

WEDNESDAY
2005
immedialu term adverse [Link]
eslge &tricb quahty contral testing far each paduet ia aloo rauied
e The dawnstheat processng md cotol testrg vat
rom product o product. bsaube
10.00

erorti
1.00
Sepration amd Isolation ef ANA fagmenla
* Treame nt DNA wth recticbion endonuclease produces
NOON

a numbe o [Link] ef vasiable lengtha.

k These cam be
besepratad though gel electrohoseia
12.00 fagmenla
-GGL GLEGibtPHOReIS Tboic
powefuzchnia,e sepratmg changed
molecWea such as protcm,iamino acida, muceabide ucleLe
acida in m electrio ield.
mole ulea have dierent migrabim
iqation Yalae.
vatas dependmg
15.00

pon ths totalcharqe, sie Cnd ahahe, wtich ea

Aepsctlon,
16.00
 The mosthcommony used imatsr Cgase, whih"
a natural palyme exhaczd from sereral speciu red
neareeds.
h
algae (g Gelidium)
Cathode) cmd anode (+) are pxesent at tuJO enda ot
C
15.00
T
the instrumento
e The NA fagmento Over cathode [Link]
placed
elecie field is' acivad 16.00

BNA fragmena Qre n

h msved
17.00

towa ds the
positiey cageaaged node end.
0
Agose haiuaseving Hetlage tragmeno move slouy V
while' the smallew moye aot ad reach the
18.00

fartthes end
NA
tgmento
Cm not be deen m visble rnd withoul

AtaininA
L Theyasestaibed witth ethidium bromide and made dit isile
Jthroughuv adiabon& a baight range coloured tbnds.
bamdaaxe cutout a agaiae gel and [Link]
LAlap is caledelution movel ot adieben
M
2
T
The
W
3
puriied
T

4 5
SS
67 8
M

9
NO
14
T W
17
T

18
F SS
then
20 21 22
M
uAec
T

23
W
24
TFSconttng
25 28
m

26
W
27
S M

29
T

1 10 11 12 13 15 16 19 30 31
 FRIDAY

xecombim nt N A
08.00 them bo donmg vectocs
attachng conm
0900

-Bioreactog -
toge ioveactors Qre the lauge contauin
ce jn w the
biochemical processes qre
100
cells
Cmd thei mediun.
ktcon eithw rooth
e batch cltue
NOON conbinuouA ue
system
aeSn batch cltuce the trnsformed recombingnt
12.00
cels Gre yatern,
aloued bo Pow in lasge bibrea tos till the
maximum passi ble Qmount f bothemical hao been
13.00

T he
fomed.
bioreactor ia emptied cleaned Cmd filled again oith
Cture Medium and inoutuo
14.00

cntinuous culture( chemostale), there is aa egula

k

dYainage melium Qnd cultuie cells fom the one sd

nd åddinien q freah medium from the ohu side
an
eon continuou ciltue system, +he cltuue cea One mauntun

phase ot pruduce
most acbive stge
biemass omd
exponenbal
lae
e
hodeied prducb
culture
fed batch fementation j8 inumeduale bethuten

Obatchcltue aystem conbnoi cultue system. Hue, the

witth fresh
continueuy Aguentialy fea
medium otbout the
Temovm
kA biDreaca provides he optimal condibion for achiev

Slithedesired produch ogtimal grawth cendibon

providng
suchas. temperatoy phabtati all vitaming o
TiorYeactor nbite chnelogy ia generay 100-lovo
cylindrical metal containe witb cuwined b bo alaa

1 2 34 5 6
cmtentsMT
IF
7 8
S

9 10
S

11 12 13
W

4
F

15 16
S

17
M
18 19
W
20
T

21
F

22
S
23
S

24
M

25
T

26
WF
27 28 29 30
 12JULY

SATURDAY

*The clture mediumcontaining au he hutrientd, salts, vitmins

08.Q0,

ec. i added alonq oith ngtim oy bomfomed cela öt

vecombinmt SNA.
The mot commly biorcactoY ig e atinring e
09.00

U
G
Stired tmKbioreactor: 10.00

usually cylindrical or with

1L.00

ut 1s Curre base o

jacililala the mixing G the Contente.

YeactoY
The atirer faitlatid mixing md oxygen availabiity E

* AJEADaivciy a com be. bubbled through the reacta 12.00

p
The bioreactorhas

y An agitatoraystem.
9
An
Afoam
tnconhol atem
gaem delireny
13.00

14.00

y A temperahue corttl system C

T
pHcontel
samplung porta( fr pesicdic wihdrawl Rhe caultise)

16.00

17.00

18.00

31 SUNDAY

AUGUST
MT
2 3 4 5 6
S

7 8
M
9
W
10
T

11 12 13
SM
14 15 16
W
17 18
F

19
SS
20 21
M
22 23
W
24
TF
25 26
SS
2728
M

29
T

30
W
31