MATERIALS AND METEODS
Followins
eat
nats wcrc ued in thb nudy
BACTERTAL STMTN; AND PLASMIDS
Ths
A,.t/i6 slrliD
a-myl6e d.ticic
purpose
ed
for rhis sludy ircluded
strain (14289),
Erc,
,acitas rdrrlis
163
(tAl) ed
a g€ne@u gin frem $e BGSC for
only. Simildly, the Ecol, st.6ins w€r. obiained Aon
rhe
Raeh
culnE cotletion al
CEMB.
VECTORS
.
pHB2ol Ghunle.xp6sion vftror)
.
pCR 2.I (TA clonins
v4rot
Obtaided from .ulturc @llecdon ar CEMB.
ENZ}A'[S AND REAGENTS
Ari chicrion .ndonrctees
ad
ihc T4-DNA tigase c@yhe, Eed in this
sMy
vec puchas.d frch tb. New Engldd
Biolabs (NEB) Cambridsc,
pobneEe wos obrained frcm the
ne production lab ar CEMB. The Hindttl
dieesled
lubda DNA siardtds
CEMB. All @genc
chemicals
!$d
in
.E
we sle pFpaFd
th€.ow
The culrurc of
by $e Eoz)me p,oduction
of this sirdy wed
fton Si8lla Bidhenicars, USA
USA. Taq D\A
pGped lion
€srh
uft
al
8ftde
or M€rck BiocheEicah, G€many.
a,cr46 sp.cic R2 ured in
49
rhis study $.6 ierated
fron t6.l
PII METER
fd
A pH nerd of EEL (Mod.l 234) \l1!! ur.d
thc
ddqbinadd of
PH
i! tti!
SEAKER
A reta.y sh'L.r
LAMIN
of'rrb [E iE$do! 3h!tc/
w
u!d.
R AIR FT.OW CAEINET
LmiM .irflow of'"rahnicho S.i.ntili.
SuPplt
rc
khoe"
us.d
Cf,NTRIFUGE
csEifue
of
"B@k'N" (Mo&l
12-21)
B
se4
SPBCTROPIIOTOMEIER
sp€{rD ploroNt r of
P.*i! EIF
Uv/vtu
0rEbd! l0)
E
tlcd lhlughotd
f,(X'D
trUME
A
nlm
bood
of'T-b.i@ Sciatific SuPpl/'t L@.
srARcH
sl!fth 3olubb A@
Mftt ot log No l-01252.1000 w
u$d a subGtale
MNNG
N-@t[yl-N{itcN-nitrco $&idifl (MNNC) bouelt &@ "si8Ea chdic.ls"
DTALYSIS MEMBRANE
Steta/Pd Mcnhae t of\petun Mdiql bd'lrtries I&"
.[ow
rbe
tui6ul6
to
!4s tNing nd.cdr stiSbl of6E KD4
50
m
E
d
tdict
MDTIIODS
MICROORGANISM
'Ite ,a.ill6 sp€.i* for ftc plod@tio! of d@yla*
babitat imhrding
oI
eil
smplc
$il, flou,
w
induti.l ed
slashy,
added lo a
w
iel.l.d
qnte
CAMB
fiom diffe@l
@llcction. O@
gu
qtr.
The
elN iub. @ lining lod st€rili*d dhiill.d
! w.Li balh al 80'C for 15 minut s AfFr lhe
oryletion of 15 oinu|s fte t61tut6 g4 innedialely @led in ic.sld waler,50d
lesl tube
of th€
M
sh.kd ed pL.ed in
splc w
1?.c for 48
pLt d our
hom.
on tbe
Ntri.trl .g8r
The €oloni€s foming
st€5ked on the n xt plsles of rhe sme
@c fotrDion.
cle
n.diu
'ftd lh* i$lat s {4
lorS-€d
dnes
The
Nbd
lo gd the
d!!at.d
**.ror.d al4'C. Cly@l st@&s veE de
20Pc for
nediu.
on lhe
pEparcd
pldd wd i.cubdqt
dEn
{@
picked up
pw cultw tid
sletj of
at
ed
io contrm
LB age Eedim
in 15% glycDl dd
&d
LePt at
-
stoage,
CIIARACIERIZATION OT BACIERIA
MorpholoSrcal
@rding b thc n
dd
lhods
physioloeical
deqib.d
popcdi4 ofrsctrldlp R2 w.F
in Bcr8.]\
Maual of svn ddic B&ldiologv
1986'.
NUTRIENT AGAR MEDITJM (t'TI-6,9)
S.No.
I
2
1.5
3
3.0
2_0
5
18
5l
investi8ated
GRAM SIAIMNG
A d$p ofdisliucd erlrd
lctcd tom
wid
rh€
ft$h.ulie
on lhe slide
M
pb..d on. cl@
\,4
@d drv
gla
slid.- B4tdir
ld.led
d
ftc
m
air dried
sd hdt
sd fd l-2 nidrB'
ix.d
an &i.d and ok€ned
positirc bod.na
v@ viola i!
udo
of
droF
rdl
wta'
l-2
$lmion for I-2 minutes Agrin whcd {ith
fl@ded with
*6
3_4
v8b€d eith mLr
'odine
wer added on thc sm€d, washed with wder
of
safteine
soludon
drcp!
Th. slid.
v4
wilh rhe h.lp of si.rili4d l@p ald nix€d in thc dtlP of
gotly to fon s sne& ll
.rys1d violer $lution
thcn it
u
the micosope in
tier
I
-2 midute
oil ilm€Eion G@-
@loE, *hjlc Srd_reeatiw blctqi.
w
pinfirh
td
in
SPORUI"ATTON
sporularioq
E ttqlcd on rhe pelri plat6 of T-3 dcdiu fd
plal6 l|@ incub6r.d at 3/C for 72 ho6 A dDp of disiilled Mrd s
pla.ed on a
clu ed dry gla$ slirlei vdy shall sired @lonv lloh T'3 plale M
TIE bocte.ial cdt@
wirh tlE help of st riliucd loop &d
rha nixcd qilb mler pl&.d or
lhe
slide
&icd rrd har fix€d. Fl@dcd th€ slid. *ith "Md&hite etdn" elurion
md renained on the
*.td utrl
Fu.h5in
hs b.d
all
stee
Renoved thc fiher papa slriPs
sd
tb. slidc
f ERMENTATTON
TECHNIQT'F-S
INOCULUM DEVELOPMEM
20
ninul6
flooded with "Cdbol
ud.i la! wtd u6l
$5shed our. Air dled the stidc .nd obs€Ned
llwaair
washed $e slid's wiilt
gen coloq hB ben wh.d oul- ILen it @
. Aftq om ninurc, Mhed
fd
pjck'd
urdd th.
thc entiE r'd @loE
nicmsF
in !d oil
25
in
bl ofm.diu M-l mntaining in 250 nl
m.!tocl . f@ |5 nirutcs at lslbs p6@-
inoculsled wilh s
conical
It
B
flsk,
*a
sldilized al
l2l'c
@lcd lo @m t mpenftE ed
lmp tull of the mioodgdiln fiom a frcsh sl&1. The fls*s l^@
incub.ted ar 37'C for l8
hoB
and agihted at 250Am n'
&
incubslor
shlls.
FERMENTATION
2% (v/O of l8
ho$
old veseralire in@ulM
ofbaclod culnE vs tdsfen d
in 50ml of fmenialion medi@ (M-9) co lin€d in 250n1
flstl.
nF flats
w€t€
ho6 dd !8it t\d at 250rpm in s condoll€d flvircm.n
incubated
!t l?'C
incubdto.
sh,kq. Aner 48 hos of ircubaioq @ncnrs ofln€ fl6kl w€E dEituScd ar
8000rpn for 15
for 48
oiNtcs,
The
$pdatot
w
u$d for th€ q@litalive ad qlatitaiive
ANAIYS$
QUALITATIVE
In qualilative ealysis,
wlls
werc nsde qith the
p.ui plar6@ aining nurridi aeo
nedim. l00d
wll
tlacr
uddq sreriliz.d condilios.
*ft
roud
cnd oflhe glss Pip.tie
oftbe sup.nata.t
E
incub6ted at 37'C for 24
jt
poNd
in th€
ho6.
ZoDc
dimctcs ir th€ plat s w@ nesu€d. Fomaiion ofzone doud tlE well showd ilE
QUANTTT.ATIVE
3, 5
DINITRO SALICYLIC ACID PREPARAIION
An altalin€ $lution of
followine
relhod.
A soluiion of 32
20
gtas
l,
5 dinitrc salicylic
gr@ of DNs
of
Nlolt
w
cid
(DNS),
M
pEpaed by ilt
sNFnded in atpoxi@t ly 400m1
in 300n1 of wter
5l
w
added drop
oa
q.ls.
wiF uder efficicnl
siring. h *6 Cdtly hdlcd
on a
Po&ssim sodi@ lartml€ (600sn,
added 10
@e si
mste rhe 6nal
qr..
*s
barh @dl
idd.d
h
volM. of 21. Th. DNs
qld glas od s:Lrd d
! .ld
slution
w
obtain d.
w
snall Ponioa md lh.n $a1er
solution
wu filtercd threud
en anFolw in rhc dt*. It m
$.bL fd
a
ldge
at
l4n
ENZYME ASSAY
Eizyhc agys fo.
&d
the rcle.sed
ceyle **
P..fom.d uin8 eluble sr!rch
redlcing sugd *€r€ 6iiDaled by
dinil,!
a
salicvlic &id (DNS)
slbstrale
esdr
AMYINSE ACTIITTY
Amyle &tivity
(196i)
6 uder.
w
d.tcmjned la.oiding to the nclhod of Fisha, drd Stein'
Enzyme lolution
ircuborat !r 3?'C for S
3, 5 dinitrc salicylic
lnl ws
mirur6- Ile @lio!
rcid. Colou de
stl,led 10
*s
lDl of l%
@ dd.loFd bv
h€alirg th. E&tart iD boiling Mter bath for 5 miNt€s, lhcn npidlv ooled
cbFd@.
Pdtin Elmq
Th. qlirrrioo valE
wvis
w
dd
teminrLd bv the additior of 2Dl of
Edwiog sugs libcnied
1o
silrch solurion
lhd
to
md
i.r
dctcmined at 550m bv sPocnoPlDtoE
(Lmbds l0).
ANfYLASE UNIT
On
myl* uil
libddr6
td*irg
suga
€quiv.t. lo l.0nc hdtos
hvdnle
uder @ndition of$sys.
MALTOSE STANDARD CURVE
Thc
diffddt
m @ stand.rd cwe B
concenteadoB of rullose
!!6orbe.
pFpaEd
silg n.llos 0 i3.0
w.e pFpd€d in
ng/ntl. Th'F
a Na-K phosphai.
bufid
ofsbovc dnutios 9@ mesur.d at 550nm sinS tboE bufld a
54
I1l€
! bldL
PROIEIN CONCTNIERATION
Pbt io @llmraldon ws detemiD.d by uinS 'Bdtlfo.d n lhod', 2pl of lh.
protein
spl.
nix€d with 2O0Fl dye in ?98 Fl of PBs (lX), 20081dt! in E00Fl PBs
CULIIJRE MEDIA
dill.dt nedi. fodulatiod sd
In it@ ShaL 0asl, nine
@yls.
t@'16.Fci€ R2
prld@iion by
llt
cmF$ton
of
r[*
u€d for d-
m.dit in Fqt
MEDIA
INGREDIENTS
MN
-t;;;-lv" Y@t Exiieci 0.5"/" 4d Nscl0 5vo (LB-nediu)
M-2
Peptorc 0.6% C&*in
Hydmlyzi. 0.4vo B€ef Exlr&l
Ycrst ErdB.l 0.3Y. ald
$eh
M-3
bM
lwldt
0.05%
|
I
M-4
2%
cacl, o.o2% Kcl
bru t s""
o
te* *ct
0 lsolo o'ItL),H?oa 2 5% and 0 5vc
198')
sovbcm t'leal 0
I
I
0 2Yo
Sovb€ Merl l 5% C@ sllich 2% Mssoi
tsto$. (G!Ww,.r sl,
| nice
0.15%
o.tr",t
-a
toi {NtLhHPoa
o 74%
'
a(
l:
[4cso,0 05"4 (DimibBli. dd Dotena.
| ,,,,,
M-5
I
Tryptone
I
M.o
Mol'*
I
lsorb*
0.08"/"
M_?
05% Slcroe 0l% D.xrin 05% CSL 4% md
3",6
(Hdilq.t al.
delt
1.5%
l9E4)
tue b@ 35"4 \r'hed btu 2% Mssol
cucl, o.le" 0d KH,Po. o r";
|
lst.Fh t%
CuCb O,OOrn6 lvtsso. 0.05./0
55
tm.Nor 0.t6% rtrd
I
C@i!
0 2%
I
M-8
CSL
M.9
4olo
I Stash
I
(vatrubi, ct
Sllth
%
2ol0 CgCI2
195)
'1-,
0.2% KHTPO4 0 l %
ed Nacl 0 04%
Ye{$ E*atd 0 loz P.flore 0.2%
ISOLAIION AND PT'RIIICATION OT THE CRUDE ENZYME
ft.
10
fm.ntcd
btolh wG cmlritueed.t 80oo rpm for 15
s
move ells. Th. crde .Eyne
satuntion)
M
4d.lloNed to
ddtueed .r
E{bslved in
agliin
se
oinut
24
s ar
nirut s 414'C
!olu.
a minimm
aMonih
in
od'r
stphatc (Ee,4
ov€Fddi at 4'C with constet slidng nE nixtuE
8ooorpm fm 20
b!fid for
800orpm for 20
stand
F*ipitaled widt
ninoca.t 4'c
ho$
of lo nM
a!
4'c-
The
*w
pdipirrrd
Phosph.le
Th. tlialvzcd
@lle€d od
bulla {!H6 9) and disl}z'd
nixtw *6
4'C ed the supemalmt ws colle.ted fo.
mtdnr8cd at
fltho puilicatio!'
PERCENTACE OF GAMYLASE PRODUCED AS INIIIaCELLUi-{R AND
EXTRACELLULA&BY,,{CItr US.IP.
10449
loonl of fcd€ntation nedid (M-9) wa ircculated
v.g.brive imculm
lrd
250
(2oZ
rpn. Aff4
48
ninul6- Tle suF@&nl
v/v) of8d.id,s sP
ssay€d for
rrslEd rbe tin6 wirn lonl of
resusp€nd€d in 4
nl
2mgld. rd.ubltqd
18
R2ina250hlndk- ltuublted
hoc, cdtN n diM
ts
{ith
w
c.nEitueed
.l
a'@vle (exlt@llule)
Na_K Phospb.t
brlld (!H
6
7000
old
tnis at 37qC
ryh fot
The @ll
9)
ho$
15
Fllei wa
Ihe pelld
B
of abov€ buffe. Lysozyne ws 5dd.d with Iinal @ndleraiior of
this at
l?.c
for
3Dinols h g.v.
56
rhc appa|2@
of8'Larin
lla6
subjeted lo enication eith micotip for 20
tj.
with
int
t.mFBtuc,
w
I niNl€.
dal of
Ai*
s.
tbe
sdc p!@s dE tib6
ws ple.d or ie
Th€ slution
soricslio4 the g€lalb
EFli.d
!pp.ti@
to @.ltol th. high
of lhe $lution wN diePpdred.
@tlitug.d, the @tl debds \re *nled doM &d th. suFmr.nt
w etad
It
for tr-
POLYACRYI IMIDE GEL ELEC'TROPIIORESIS
SDS polt?crybDde gel
12%
w€ls
Mlving
wE
sd
smpls
of ihc
and 5% sla.ling 8.1 of
vlshed wilh
i! lx sptc
@frit
.tdtophoGis
1.5m
bufld, T}!. spLt
*6
h ! niri
Pdfomed
thich* Er u.d
of crude
loaded
6
it
the g€l
3lop9€d
botiom. Th. gel w6
ltd
a voliaSe of 8OV
And polvn dztiotr
oz}de sd BSA
bufl4 with ber shoct in boiling H,O fm 5 ditrut
wD
kepr
g€l aPpalatu
s
wt
Abo*
consllnl
diselved
30
d
of th.
The aPplicrtion
*ncn lhe trekl.8 dve @h.d ro aboul V4 irch ton rhe
cooMi€ s Ell s
3ilv.r slained io delt t the prct€in bdds
SILI'ER STATNING
silvd slainins w @fud
fix.d b
iin€s.
!
out rccodins
b
fixing $lution for 3H0 minut6. lt
Thcn it
qs
*6
Ili.
sel
w6
Mshed with dislillad wter
l*o
Bl@D,
d
al
'
(1987)
lr€ated with 0.02% NarSrO3 solulion for 2-5 minu16
shalilg. An6 sL,hinS Arin wilh distil.d wtd, it B
sled
*ilh
in 0 lv"AeNOr
Sentlc
eluli@'
Aier lo-20 ninut4, thc silvcr niidte solutior ws Emoved foltowed bv wahi,8 sittl
di$ild wt
r, Aesh &vcloping slulion
wad€qul.. Mditiod
of 5nl of
2
3M
s
c'tic
added agi|.Ld slovly
atil
acid slopped the @clion.
57
b.Dd irteNity
CEEMICAL MUTACENESIS
l8 hous old vegelativ. i!@ulm of ra.l/16
5tu1
of
nediu
LB
mt
ircd in a culiw tub.. Ir
E&hed to about L?- l.8 i...
I
x
2
ho6
i!
Esp&tively)
gently
M
suFnendl
vs
E
disc&ded. Cclls
wft
wirh
re&n nl. TIF neairn€nt
s
i!
OD
!t
600
i.e. with
000rPb
w
5fu1 of MNNG (N_
q<l incubatcd fo! I hor .nd
fd
esNpended in
I
label.d for
find conentqltion 375P8/nl.
l0
lhl
nh
to
Fltd
dE
ells.
2
The
of the mcdium LB As6ir
dom sinilu Flced@ wE dorc eith 2
siven in tum€ lDod
.lMinjM foil. nE dpqid@t
e.h) two wrc
@h reaitndl t@ th. siet
shon inteNais
d@ al ld
cdtiftealion foUowcd by esLtFsion
hous
.dd.d
wra)
sh*oi ata
how.t l?'c. ccoritugni@
uril th.
$d oib6 two for @!irol,
{?.5mg/ml E6hly pep6red in disiilled
vd.
lraisf.ded (l%v/v) in
incuhdql
6 epFDdods (1Dl in
n.thyl-N-nitrGN-nitroe guddiE)
The cppodo.fs
w
m
1o"cft/nl.
AboE @lk R@ lako
rrst'not (l &
sr'. R2
ed
etpendorf! w€E
ondwr€d in dr(l dE
ro th€
mpp.d
lidt @iriviiv of
MNNG.
CELL SURVIVAL
cqlls
of Bacils
sp. R2 at a
l75t€/nl dos of MNNG foi I &
2
d.sity of lrlor cMnl
how
The cell suspdso.
*w
ch,llengcd
wil!
w!
dihied uplo
lo'
rin6. 50dof ehdilution*6pla&donlh.nuEidlage+sttrchplal6. Ealdilution
@
sprad on th@ plates, Plates
apFd.d
w
6d trssl4,Ed
*et
@uled for cell swivallo th€
incubtl€d at
M ors
nutiol m.dim sbtis
58
l7'C for 24 hoN.
foming
cl4
6rc3 wE
Colonies
pickcd up
SCREENING OF MTIIANTS
Elq.n mulanb
vw
s
wcE slrencd qudit rively
9.
The
tine of incubaliotr
el€ded non
well
M
6 q@rit tivcly. Ihc cultft mediM
ho6
48
hoB lqtrnol of MNNG
2
qt
Th*
mut3nb
w
again M_
3?'C.
ENZYME PURIflCATION
USING GEL
FILIERAIION
Eutme puificalon wN do@ ar . t
supedatal of ih.
qntw
thli peipiat d at
di$hd
in
!
brolh
80%
wd
treatcd
w
amoniu
h
s6tll volMe of lobM
bufld
phoslhale
s€
buffd
S.ph!d* bufrd. 'IlE
6tlm
ws c(i1mlenr€d wiih lo
ws
t.Da
washed
S€phadex
buf€r.
PHT
o€
|hc
ficn applied
to a
a Gcphldq)
M
b@ eqlilibedlcd sith lonM
*ith $e .quilibFiion bufcr' md crule
The
*6
collm
.quilihdtid bufiq, dd prciN 9w
elurcd
ir
l50d in't
bad
bd.quilibd'd
combincd and
ulm6lterEtion
tbe
fom of turile tetions
pml.d oln in fiv. diffemt p.als
Bilg qtri@n
30
ed chet
d
wilh
idtially wlshed with l00nl of dt'
a. a flow
of25nlh'. FtutioN ofl2.5hv30nin w@ collected in.eh lub.- Ih. etive
m.
pbi'i!
filts h n dim dlE filt 6tion cell Th. @rederd'
pur or a @lu@ or selhldex (G-?5-l2o), of
lomM
siLlPhde, drd the &z.tion
Thc retotale
of s.ph.da{ (c'?5-l2o {l0tDa{0tDal) rh't hld
@llm
The .entritugrl
salslioq ws coll*ted Th. praiPilatcs fom.d w.e
couse of 16 hous againsl 50 vol ofthe
s
oFmtd b.bw aqc.
ud
nr'
tactroro
cotcflteEted
o. sDs PAGE aner dzJmc
bv
.sv-
USING ION EXCEANGE RESIN
To c@fim ihe .tDvc puifieriol! crude enzto€
r.npdatr
below 4oc. The
enlii€d
*6
puili.d ag.in at
supcdaitnt of rh. cutr@ bmlh
59
w
a
har'd {iih
smoniM
s'nphale,
&d
Th. Frcipitat* fom€d
pH ?,5 or pl 1's,
dd
50 vol of lhe sme
lhe
the
r*
t&tid
dislv.d in. 3D![ volM. of l00bM
$lltion
(carion-qcbdg.O ih.t
?-6. Thc
@llm ws
ellule
pH?.6
firr
coll€cl€d
Pboslblr. buffs
dialy4d twie over rh. @uM of
16
Loc .g!i!sl
equilitdted with l00ntv1 P-l I buffs or pH
tcn
llc
eqoilibtslion bufrd, .ttd the mn-adsorbcd
had
b€n.qfibEt
lind efdidt
o3756vnir FE rios of
flirc
dtzfilteFtioo (atrtriconJo; 30,000 cutoffi Ani@4
M
loa.Ld. The co.ccntol.
ws initially wh€d sith 9008r
*ith 350E
had
eEsh.d wilh
crude potein, con@ntaied by
Mdt @
M
w
bufiei. The etentd. ws! th€n applied to 5 .ollm ot phosphate
eIdG
Dmvds,
th.t Pt€ciPiHed at 80% sturation,
d
put
o
a
with l00nl\4 P-l I butrd pE?-6 Thc @lum
or rhe .quilibnlion buffd,
of o to lM KCI in fie
5.6Du15bin
@lum of ?-l I photphde_
tr4
ed
tr.
@[61!d
th€
poteia wF cluted
buffet, at a flow ratc
tilF
lhe st
tt of
of
the glddic.t.
.sv ald @rcnlcdl.d bv
ulbaliltdd,on or a eniricor 30. Tlc concoteote *s dialv*d ovmigltl egdmt
loonM P-ll (!ll ?.6), ft. EsdtinS Et dsre rc !$d.tclsiY€lv for irrhd
qFitu ssdE frEl pEplratiotrofth. FFfi€d e.ztre Th. &tive fractiotr lN@ @obined
an
t
@zlde
ENZYME ASSAY
d-Amyle etivity
@noined
ls
w
a1
3?"C in
6
2ntl Eelion
nixnw rhal
of a l.oq. (*r/wl) $luti6 of eluble sr,rch (fron lot to; M.ak) in
0.02M pho+hal€ buff.r pH6.9
rcd@ing
rcutiftly measwd
swd fomed
ed lml
rc n6u€d
of a suilallv dilut d
sldion
by th. dinidosalicvlicacid
SLiI! l96l; Mitlq, 1959). Ot€ @il of lh.
GDzyme
60
a.livily
a
ofdrrm.
Th€
pocedw (Fishei' ed
dclined
a
rh.
Doul of
pbtein ilEt prcd@d tmg of Ed@i4 sugd
s naltxe
in 3
ninut r
undct th. @ndinom
ELECTROPHORETIC ANALYSIS OR ZYMOGRAPEY
sodiM dodayl sulphate'Polyarylmide gel daLophocis (SDS-PAGD
done
senrillly
&rylmide,
?O
as
dceribed by
Lmli,
(1970),
by 5Om, l-5nn thiclnBt,
i.
ed wples w@
Fplica plat
for
.
@yl&se aciviry
tun rcnF.atre'
sls een $ cl6
M
Moleuls @3
low-@s€
nolFuls
(9?.4kDa),
lelm
trlsb
bhibitd
mvle b
Thc sLb 8el offq SDS-PAGE wd lad or rh€
svenl hous.t
slab g€ls
eplio
The b&d of prctein that
il
wirh
wd doG
sh.d
wd
ed
leri
ovd
ss@iated with
d&r blue backgrcud on th. Eplie sh4t
@ne on a
Elimsted by SDS'PAGE
IlA
sbincd fo. prot
with a€rr shel @trt inin! starch (sigm)
Mda[y s desdib.d by Hrladad al., t95,
6
fom of slab e€h (10% [s/vol]
rhe
Col)ltNi. brilli4t blw R-250. Acriviry sllining of
M
(loeldv/loll ervldide 8'l) witb
stard4& (BicRad), shich iiclud€d Phosphoryle b
albumin (66.2kDa), ovslbMin (45kDa). carbonic envdrae (31tDa),
(21 .skDa). and
Ly$zym€ (l4 4kD.)
EXTNACIION OF CENOMIC DNA FROM BACTERIA
Tord gmmic DNA
sltai!,
6i.g th.
mcthod
M
(c
az.,il6 colo.y
spp.ndix). Allowed c.lb to sre*
O.Do @hcd O-8. Thc eU! wE
pelLt
rc sh.d
with
r!r,,lir
158
(BGsc
.lesibed by KJomt.d, d al. 1983; Sdito, ed Miua
ovemisbr cultu€ frcm a sinel€
D€dim
isold.d from thc ,acttl6
hd.n
mh $luti@
d
!t
@
!t
dilnLd (lrl00)
37'C in
?,000
ii
& in ub.ror
6l
1963
50nl of LB
shaka
+m tur l0 bit\ d 4'C
floo'nM N.cl, lonr{ TneHcl (!H
lAl)
7 9),
util
@d the
100ftM
EDTAI ard Buspend.d
i!
EsusFDr'on solulion
I
forl bour.
To this odded 6,25
ootnMT.isHCl(!H7.9), 2%sDsl ro bRlk
60FC
til
n ba,m cl@, rho ir
w
nl of
!4w$ l,ts *s @tully
(2411)
aspiFted otn
.bslule ethdol. Ana aitiryine, il
oncdt€niion
w
PREPARATION
$tim.Ld visually
A
of 025b8/nl
dd
duact d
nixtw,
sd
one timc in
€q!.ll volme
Mov€ th. tr@s of
to
piPp€t€d with 2,5 tim€s
th.
ieub6Ld.1
tm rin s with cqutll volw. of
w €sPddd
agoiost
M
Phenol. Th.
rclme of ie'@ld
Lw
2nl ofHro (DNA* fre,
'n
0.8% asarce gel
tshds/titd/
dd
its
r.l 3i.nd.rds
OI PLASMID DNA
Pl6mid DNA
1979).
(!tl
dou celk. Th. spcNion
&d altoclaved). TIE DNA *&s ch4ked on !
E.C
TrisHCl
lonlt
Lysis sluiion [loomMNdcl,
ph.rcvchlNfondleuyhl@hol (25:24:l) mixtw, od
sith Chlomfomn$mylolcohol
NaCl,
! ftal oc.nrcdiio!
7,9), loobl,t EDTAI. Addcd Ly$zync io
incubaled at 37"C
050nll
*6
prpaed by
sinsle bacteiiat @lony
$. sltalift
lysis method (Binboin
dd
Dolv,
w6 uFd io inocul.t€ 5ml ot LB nediw
Gee
dpp.tdix), onhining th. appopnate elibiotic 4d ercm al 37'C wilh vig@u shddng
fd
12-16
ho6 (ovmisbt)-
shined
nicrccdcituse $bc (.pFndorD
l.5nl of the d.didl culrw inro ! l sht
ed c€itiitus.d
D@eled the supemrant md rcsupod.d the
at 14,000 rpm (l4K) for
I nitut
Fll.t in 200d of ie{old. .ell
dusFBion elulion (z5nMTrisHcl{pH8.0}, IoDMEDTA), incubdql for 5dhut
on i@.nd
tho
added 2o0rrl
Mixed rhe conte s
cld
e!.nl
ofAB
.
y pEp€r€d ceU ttsis
elurion
times by inv€ning fie tub€ g6dy,
(l
lotil
s
vosDs, o2MN.CU
the solulioh
b.cmc
.nd ad.led 20qrl of ie{old N.utdizriod $luiion (1.32M PolrsiM A@Lt
{pH4.8}. Mix€d by ev.td iNdioN dd incuborql @
62
i€
for l0
oilut6
The
ssp.Dion
rc
sup.tuiant orce, wirh cqurll
on@ wirh
6
CblorcforE
minures, follow€d by
Hhdol
vol@
2.5
lim6
minut€s
(4'!C), Exl@r.d lle clcu
of ph@vchlotufom^stmylal@hol nixnlE
edyld@hd
rbd p@ipira&d wirn
with 70%
fo. l5
harested at 14,000 rpn
to
movc
rdu.
c{nifuatio..t
and aA.t aiFd.ying,
of
14,000
ph@ol
iaold
rffi.
dd
T[e cl@ a4@a phts
sbellde e0ts@l
ar - 20pC
rpn for l5 minu16. nE p€ll€l
for 15
va rinsd
ws sspended in 25Fl of nEle@ Ii.e w.tq.
JIT'I'IIS
TRANSFIORMAI'ION OF COMPETENT
CELI-S
'4CIZIUS
COMPEIENT CELL FORM AT \ON \'A CILL US)
Cotup€l.nt
elh
of
r.rrrt{ir wF
Hom(1990). 2nrof spiI c'nnE
w
*irh 200rpn sbabng The follooiig
Spil
!d
@nrinued
a dsnb€d
miDuc
supmaiali
moming rhc culnE
phe (i..; m lignifiMt
cultN (1& ) w nix.d
&d enriituged .t
@
ed
Vand.r
i!@ulsLd ov€nisltAm at$h@lonyar l?'C
at
9a
diluted eith
l8r
iish
eved
ed
(80009) or 7K
ihe FnaioioS
change
i!
@ll de@ly ovd 2G
lhs ir.8. witb l80rl of pr4m€d Spill dd
continEd incubalior at 3?oC for addiriord 90 minut
l0
by Cutling
iMhodon !l l7'C with vi8oreB shaking (180r!m)- whorbc 6r.
of eU Smvrh l@hed s&rio.rry
30 min), th€
pEpdFd
-
The
.ulte G
for 15 minulA
lt
wa cNtully de@led.
kepr on icc for
0-4
"C. l6nl
of
The cell pellel was
86tly EsFnded in 16n of th. sv.d suFmatar with arldition of 2trrl of stdile
abelute
ely@l
or 4ml of $dile 50% sly@bl. Aliqrcb of @mpetdt
*w tansfm.d
1.0 drl)
irro srqilc tub.s,
liead opidly
al-80pc-
NOTE:
Ia l8 ml
suptur fiq
2
mlof(abaolur.) 8lyerel
6l
ells (0.2,0.J &
in liquid niirog@ and stor€d
.
Tubc! tutor
ad
cpFndorfs
lNd
k.pt cold
udq rleril. @ndiio$ d
l€a.r
fq
e
TRANSFORMATION
ft€ omFtenl
cells w€rs thaw.d r.pidly by
inmding frcrcn tub.s in s 37'C
water balh. 200p1 of Spilll was added io 200!l thawd celb, mned genily wilh 10td
DNA elution (0.5-5pg) md thc
for
I hou
nixtm
m
wiLh shaldns
mixie
w
incubet d al 30oC for 90 ninutes or l1"C
(l50am) in a $raiory incubalor shak€r. Ibe tlsfomaiioo
plal€d or LB 5g& plat
e
wiih el.dive &libiolics,
TRANSEORMAT]ON OF PLASMID DNA INTO ACOT'
COMPETENT
CILL FORM TION (E.ori)
e.ot onpelan alh \r@ pr.p.Fd
Hdrld,
198q
@lony
in 2L
1983)- 2
of,'coli.
flak
2
nl
of
u
nl ofllis sd
for abour 3
wirh the C!Cl, hethod
owniSht LB c!lt@
culnG
hoE erd 30 s
\'@ ituulst
O.Dm |@hed at 0,,1-4.5. The cultw wa! k
enrritugrtiotr
dt
4000rpn for l0
niru|s
d in
l?.c, 200rph
at
F
04'C
at
*a
in
on icc for
in a
(Subek,
in@ul.led
liob
a
et al.,
iq!
200d oft6h LB culrN
r gtFtory
l0
shakd
unil fie
minules md peue!.d by
JA'l4 rctor od tub6 (pfc@l.d)
w@ lruh€d twice by Esspoding ed rccotrifugation with 20ml ie<old o.lM CaCl,
solulioq
50%
dd
g.nlly
@ susp.nded in
cly@Dl. 100!l of @h
pFchill€d nictutuge lubes.
4nl
ic{old
suspcnsion of
0.
lM Caclr, l.5Toglyccol i.er ] 2nl of
conpetot cells w6 i.mfomed to a sterile
Th€ comp.r.nr @lls wer. stor€d dirccdy at
-80'C.
TRANSFORMAIION
Ior trsrsfolmtio4 plMid DNA
ircuhsted on
ie
for 30
nh.!d
b
han shek
mixcd wi|h l00pl @mpetfrt
sa givo tt a2"C in a Mre.
ells
and
ba0! for 9{)
5@n&, ed or
ie
for I minut . aoq{ of LB Fcdim
'6
&ld.d
sd
imublr€d !r 3?'C
Itr.rory incubotor shal.r for 30-60 Din e5, l5qrl oftelfonaiion mixtr 9E pbtcd
onto
d
agd LB
pl.t
with
ad
(Mrtin
appDpriat stibioiics i.e;
lui!-B.rt ri
37C for ore d.y on
strrch
d
(LB)
.gd
plates
tdfod€d ells w@ crcM.t
$pPlclMt d with l9n(qi/toD
(25|re/ml).
AMPLITTCAITON AND ST,QUENCING OF DNA
fd lh.
mpli8c.tion of
lp!reFiol€ Egios bctw@ speifc sitd h thc gelomic DNA 'nE
F* ""qld6
shon oligorelotid€ equmes
wE
d€siened
CCCAACCnCCGITCACTCITCAACTTOTTT1CAl.
Hf?o,
t-
pi'net HRPO
5T
sEQr, tr
CCCAACCIICCGCATCCT"ICCAGCCTATOTT'TC.]',
TGATTTGTATTCACTCTGCCiAC-I', SEQ2, 5ITCCCTCrCATTTTTATT(CTG3
sEQl.
SEQ4,
5'-CACGCGGTCATCAATCATAC.S"
.
5L
TAGTGCCTCCACAOATGCTCI', SEQ5, 5ICCCCCAOTGCT'ITATTTCAA.]''
CCTCCCTTTGATTACCTCCT-I'. 11
om@ially (lDT).
PCR
PTC200, 100), with each
Aa.il6 er,t/,s
m
pind
y wE
I
DNA svnihsid
lheffil
cvcLr (ABI, 2700'
pEporcd
p€.fom.d with a DNA
on
(l0pmolctFl), plus cBnomic DNA (50l00ns), fton
168, (95qc for smin,
cycl6 dd zqc tor
94'c loi I oin, 55'C for lmin,
lonir). Ile @don mirE
ph6phai6, 25nM MgCl:, 2.5 uirs of Tlq
prcduction Lab)
sEQ7,
5'-GACAGGCGTTTAACGATCjCA-3',
SEQ6,
dd lx Td$HClbufr{
2nin fot 15
ontained 200FM d€oxv'nEl@lid. Ei
polynew (CAMB, Rcstliction
(!H8.8) in
65
?2qC for
a
r&tior !olM.
wFe
of 20d- Prcd@is
wG Frif.d witl r
of PCR
f.. cldios .rd
0n
sl., (1986), by BioS
pcrfotutd
ly
w
crti! ldtir.tio!
tlF didcoxy
3lr.nd of lh. DNA of Aa.i?(t
3.$dcd !n r TA cloiDs dd
!trdr5tu
B
ut d
EFttbd
ofsnih,a
fluot!$4t t@iDto6 .!d e.doolncd DNA !.qu.nc.i (Mod.l
ll00i ABI, PRISM). Sir'8L
B
lhtu
.u!-cloning.
B
S.qwi.8
Frdfc.li@ tit (QIAOEN), lDd
PCR Foduct
r!
&nc uirg
Gxl6siotr
onlit|c ChEE$
d
rctl& ddyL$ @lt
LB+Sl!rc! pbl!,
!!d
gpn
coinF
a
pre8ru.
NUCLEOTIDE SEQUENCE
TIF uEl6ti& lcqueacc &L, rbocld 98% bo@losy witl
@yhe so., ! lorch i.gnding qE B.
(EC
&c/lB n,lli6
3r.l.l).
PNEPARATTON OF DIALYSIS TTJEIIIC
ltc
B
rubins
cul
i
Dirurg ir I lasc volue of
ritr!.d
lbo$ur
y in
B.for!
pies
2%
e,
hrodlc ds fubilg wirb
io
cooHidl l@gll (lc2ocdr. Boil.d for l0
A8sin boilils
@l .d
rb. rubiDs
slo6.
of
$dim bic.ftondc dd
dinil.d HO.
lbM EDTA, Allored
sbD6d.
o
N
(s@btlolq
cr al
EDTA. Ttt n$ing
M
dolc for l0 minulB i! 0.001M
3rd!d rr 4"C. B.
w ruu
l!
3w th.l th.
i$id. .rd o|' *itb
@ld
tubin8
d
it alsf!
D.tllo. Alsry3
l5a9)
PROTEIN TRAIISAI,oTTING
PnDPaRATION OF P'OLWTNYLIDEIIE TLUORIDE
(r1'Iln
MEMBnANE
(0lrr)
a im.n d in 10094 tutuel for .
fcw rccood4 undl tlc eott t!@b.o. *.! ltlldEot I! EcO4l, it wts
iEEcdi.t ly. Wcn d lmbtu E r.Bft.Ed !o . t!*l co rininS Etaf6 b!fia d
M6bnF
w
clr of d!.t iiz. of gcl. Ii
6
water' Incubat d in Towbin butr€r
s
mdbtuc @ It
bd Mt
6ily
it
is
.quilib6ted ('2-3 minut€d rhe membme
bulfr uril
flosting on th. surface of thc
equilibrar.d it @uld tE
h6
ulil
completely €quilibdt
$bturged irro lbe
dy to bind pbEins in
sy
aqEu eltiion
d.
va
Affer it
t!.
Al this loitt,
blonine appl'@riot And th.
n@bEE
$ill fom
wheE th€
d w'th bufer, do nor allow it
b
dry (while spols
ment{ae is dry). Potein will not bind to th. dtied ndbrd€ ed dry
spols
qill
nol
Fset in .q@ut el!|ion,
TR NSBT,OTIING
I}.
fotlo*ing prcto@l
*s
for use with a
for holt prci.iu, ue a
trmbl4
prcto.ol
i!
stDutd be
thc
d@ .nd
rcBhuc
Towbin bufter with neth&ol
tF
weding
of
si.kl6.
eslern bloting
lsfd
6@bre
s6tior Mr].
@ mehbrde for
the tin6
(5
I hoE rnd
z@
lO
m
n nbm€ slain.
lrd
lhft
rct1age no1
wt .-
(M.OH).
P\IDF
Shinine
D.slsining
folo*itrg lLe
should be no bubbld
also soaked in Towbin
shi
dry
buffa
4
tdsf€r etl
w
imb'4 B
donc for
wd for l0l5
rinscd
t niru&s wilL
ninutes, or
uiil
ba.kgrewd is light blE, with PVDF dcsbin solurion Avoid eetc acid in the ttain
d€s&i.,
6
it might
clN
in
boF the t lvolts for Pdein
oinut6- An r tEsfq,
ninut s @h) wilh distill.d
PVDF CBB R-250
surE
md bloni!8 pdp€ts on
@ *l- CuMl B
Ttffi-
s wl
Meobl@
b€tq@ lhe nembtr6e ud the gel. Blotijng pdpq
wlDle sysreD i.e; eel
the
lslk blol appostB !@h d
&d
blctrge ofthc anino teEinB Pl&.d th. air&ied blot in
67
MhbIffi slFlld b€ dry in air .nd
ndb@e iGtocli@ baNal
bloftad
Cat0,
&s1aining (PVDF
(BIO-RAD), C.1 4 152{184
d r polyvilylid@ diAldid. mmbtue
liicl
had
IE
Pnkin *qFrcing
spl. B
enzvn
(Poerb; P.rkin-Elm6, Foslr citv.
ben Ell€n wih nedlml. The
diFt miEl
s€q@@ of tltc
potcin wc mr y€. detemjned.
ROTOFOR
Isoel4tric points of diflernt
pot iN
weF foud with the help of Rotofor sF1eh
(BIO-RAD) lollowing lhe Rolofor R eystcm lnslruclion
IMud
according
lo dE
lab
SAMPI,E TRDPARATION
Sdpl6
9ft
ilesalt€d by dialFis. In
lh. crud. smpl€ 02% ls-Octyl
ptEdy
poly (oxtrthyltr) elleot oCEPAL}0.1% Ch4s 13- (3-Cholamidotopyl)
din lhylamonioll-Prcpmesulfonic Acid (i,ci no!
elubility or the feus€d pm&i6)), shM DTT
(1
io.i. detqgdt
,+Dilhaofieitol ), 0 l% cly€ine od
Anpholin€ (b naintain the solubility ofth. @plc ed prcvid. bufqirg).
SETTING
TJT
maintain rh.
l*
dd.d
FOR A ROIOTOR RUN
Anode md €thode ele.tolt'le
chMb.s
were fitst
Ndbl€d.
Prior
b 6edbly,
th. ion€xchang€ m€nblffi w€e equilibdt d ov@ighl in th. apptopriate ele.trollt
slulion i,e; in o.lM NaOH &d o,lMItrPO3 for eion ard @tio! 4ch,4p m.mbtu6
rlsp.clivcly. 'IAa elet$d6 w.c .s*nblcd. F@sing chdbd
spl6 *@
F@rionarion
lmded.
@
t
ading pon!
wc sal.d.
sbned. Mdimm sFcili.d
68
An bubblE
ops ingDatderss
Fdcrio$ V@ @[dt€d aftcr rh. @nplction of Rolofor
6
M.
UeFIld
re
fo.rhe
and
moecd,
ell wE Ft
SIIDDIRECTED MTITAGEIIESIS
To
pr.pe singl€-nutdt
two muiag.dc
pnnd lr@
in
d oP.dor
synlhdiz.d
r€giot of myE qith +5G+A nutaiion'
6 follo$
usirg oiline Stralagen
snse
B8{r 5 grgangrSekattt@d g@ gcAn@!@tlclccrgtacel
eng84a 5'$gr.ea.eeag@ftgneTgctt €tn@trarc!@tcrc3'
Si|.nietcd nut4edis vs
done
followbS th. S!!&goe XL iilrtudion
tmu.l
with
lligltmodin@tioN. Firl slepvs
.
Pl6mid pr€pdari@ i-e.,8.ne h
.
T.mperalde cycline i.e., to
plffiid wi$
d.mtw
th€
tar8.r sii€ for mutation
plMid &d m€al
the oligonwl@tide
prin€$ containing lhe d€sied nutation Ning quck chdg€ XL sitnire.t d
dultgddis ldt alonryilh t mPld. DNA Prin6 @d H,O
Using thc
rc!-
stdd{bpla.i4 a.tion of ptu Tubo DNA polvlffie, €xld'on
ard
imtpoddng
.
The &recl
lhe murag.dc
prin6, tick d circde stredr w.E
Pap€t d.
nulalio! in pcR2,l clone (i4ptate), catrvirS dvE, ws ceicr' oui
wi0r PCR mplificstion prcsmne of
95t fot lnin (fiBt deDalMiion)'
25
cycl* cosistirs of 95'c for 50s.. (d.@t@tion), 60'c for 50s (enn'6ling)'
6E
c fot lonii
The ncn scp
(eloDsation)
*s
ed
t&( cv.t. at 68'c
digenion eirh
Dp,/ io drgd
fd Tnin (fiuins) ed 4'c
tbe meihylar€d, mn Du|,r.d
pqcltal DNA templare. So id th. PCR prodE! Dp,l
appDxirutely
I hod dd
45 s4 al
37t.
for
s
a<lded for
T@1ed DNA (PcR Poduct)
ws
on nqr d.y DNA
To the podDct, add€d 75%
RT iftubaliotr
v6
w6
prccipilalion
isopFpsol. Mi&d rell fd 20s
givcn for at
ldt
Sup€mtart
dd
pellet
m
by
piFttjlg
s
.d
20din. spin doM th. prodDd at 14000ryrn
E
fo.20mjr' .nd at RT- SUF@ra
pelLt snd v.sbing
ispDp&ol which is
done drcugh
q@tully Mor€d withoul disruling lhc
doE ag.i, with
?5vo
w4.gai! cGnrly d.cet
d
i$pnp.nol for
ed !n
5min at 14000
dri€d for dt le.s1
4m.
lonin at RT
wa reswFnded in I IrO.
TrsfomEtion i.e.. uesfods uE circuld. nicked dsDNA into XLlo€old
ulE@nper€nt cells. Ai
R6lsFded
ie
peu€i
w
fii!
mixcd
sr.p Toplo @mpetent
*ith
for al l.a.t 3omin. Heal sh@k
@mpetcnl
s
ells
els dd
wc
iha*ld !t RT.
ilcubatiotr
gim d 42t fo 30-a!$.,
6
aad
dorc on
lo ii add.d
Soc (n€dim). Incubaio. ws giv.n at 37t sldory ircubald shsld for
appbximtety 2 hoN dd
30
s
thd
sprcading
eEoed
after
plenid isolslion.
dorc on LB+sr.Eh+Ku or
(fq contro!). Mulantr
ADp plal€s and LB+X-GaI+IPTG+Anp
v4
M
r€sis1a.t 1o
Doubl€ $reded plasnid DNA
tpa]
w6 ued
for atl seq@cinc @tions again lsine didexy chain tqni@tion m.thod of
Smiih et al.. 1986 by uinS
flldeat
scqEM (Mod.l 3l00:All, PRISM).
inro
rmimloB &d
Mut nts
\,@
e
aulon
sub.lo.Ed
ad
ton
pCR2.l
pHB2ol ald rnEfomcd inb E..oli, Arcills &.brilis aalletn B@illu
70
DNA
sp.