BACTERI

ALTRANSFORMATI
ON
Ai
m:Topr
epar
ecompet
entcel
l
sandt
ransf
orm pl
asmi
dDNA.
Introduction:
Bact er
ialtransformat i
oni sapr ocesswhi chinvol
vesgenetical t
erat i
onofbact er i
aby
i
ncor porationandst able, expressionofaf or
eigngeneticmat eri
al from t heenv ir
onmentor
surroundingmedi um.Si nceDNAi sav eryhydrophobicmolecul e,itwi l
l notnormal l
ypass
throughabact erialcellmembr ane.Inordert ouptaketheforeignDNA, thebact erialcel
lsmust
fir
stbemadecompet ent .Compet enceist heabil
i
tyofacel ltot akeupext r
acellularDNAf r
om
i
t senvironment .Ther ear edi ff
erentmet hodsofcar ryi
ngoutt ransf ormat ion,e.g.chemi cal
transformat i
on, electr
opor ati
on,genegun, li
posomemedi atedt ransf erandmi croinject
ion.
Chemi cal tr
ansfor mationi ncl
udest heusageofCal ci
um chloride( CaCl 2).Thismodeof
transformat i
oni seasyt oper form andr equiresminimum equi pment .
Principl
e:
Fort hei
ncorpor
ati
onofplasmi di
ntoacel l
,bacteri
amustf i
rstbemade“ competent
” .This
processincl
udesthetreat
mentofcel l
swi t
hbivalentcal
cium ionsinice-
coldcondit
ion.Asa
resultsmall
poresareformedont hecellmembr ane,whichmakesi tper
meable.Thepl asmid
DNAmayadher etothesurfaceofthecellandupt akei
smedi atedbyapul sedheatshockat
42°C.Ar api
dchil
li
ngsteponi ceensurestheclosureofthepor es.Thesecell
sareallowedt o
propagateandselect
ionoftransf
ormant scanbedonebygr owi ngthecell
sonasel ecti
vemedia
whi chwil
lal
lowonlytheplasmidcontaini
ngcellstogrow.
Plasmi dsar eextr
achr omosomal DNAel ementcapabl eofi ndependentr eplicati
oni nsidea
suitablehost .
Plasmi dsencodeawi dev ari
etyofgenes, includingt hoser equiredf orant imicrobialresistance.
Thesegenesactassel ectivemar kerswhenat r
ansf ormationexper imenti scar r
iedout .TheE.
coliplasmi dpUC19encodesagenet hatcanbeusedasasel ectablemar kerdur i
nga
tr
ansf ormat i
onexper iment .pUC19hasampi cill
inresistancemar kert hatenabl esonl y
tr
ansf ormedcel l
st ogr owonLB–Ampi ci
ll
inpl ates.Tr ansformant s, thushav ingtheabi lityto
growonampi cil
l
inpl atescanbesel ect ed.Thi spr ocessofdi rectsel ecti
onofr ecombi nant sis
call
edi nserti
onal-
inact iv
ation.pUC19al socar riestheNt erminal codi ngsequencef orβ-
galactosidaseoft hel acoper on.TheE.col ihostst rainhasadel etionatt heami not ermi nalend
oftheLacZgene, whi chcodesf orβ- galact osidase.WhenpUC19i st ransformedi ntothe
compet enthostcells, t
het r
uncatedpr oduct sf rom bot hcompl ementeachot herandasar esult
enzy mat i
call
yactiveβ- galactosi
dasei spr oduced.Thi siscal l
edα- compl ement ation.The
tr
anf ormant stur
nbl ueonX- galandI PTGcont aini
ngpl atesduet ot hepr oduct i
onofβ-
galactosidase.X-gal isthechr omogeni csubst rateofβ- galactosidaseandI PTGact sast he
gratuitousinducerfort heexpr essionoft hisenzy me.
Requi rement s:
Ampi ci
ll
in
Luri
aBer tani (LB)Br ot handagar
E.col iHost
Plasmi dDNA
1M Cal cium chl or i
de( St eri
le)
X-Gal (
E.col iencodesaβ- galactosidaset hathydroly
zest hedi sacchar i
delactoseint
othe
monosacchar i
desgl ucoseandgal actose.Theact ivi
tyoft heenzy mecanbeassay edwit
ha
chromogeni csubst r
at esuchasX- gal(bromo- 4-
chloro-3-i
ndoly lβ-D-gal
actosi
de),whichi
s
conv ertedbyβgal act osidasei ntoani nsol ubl
edensebl uecompound.
IPTG( I
PTG( isopr opy l-P-D-thiogalactoside)isanonf erment ableanal ogoflactosethat
i
nact ivatest hel acZr epr essorandt hereforeinducest r
anscr i
ptionoft helacoperon.
CollectionTubes, Pol y propy l
ene( 2.0ml )
Coni calflask, Measur i
ngcy li
nder ,Beaker
Micr opipettes, Tips, 50ml Cent r
ifugeTubes, Drybath(42° C),37° CIncubator,
37°CShaker,
Cent rif
uge, UVTr ansi lluminat or
, Crushedi ce,Ster
il
edoubl edistil
ledwater,Ster
il
eloopand
spreader .

Met hod:
Prepare0. 1M byaddi ng40ml of1M ster
ileCalci
um chlori
det o360mLofst er
il
edisti
ll
edwat er
.
Priortothepr eparati
onofcompet entcel
ls,pre-
chi
llt
het ubes,0.1M Calci
um chlor
idesolut
ion
andcent rifugetubes.
Setthecent ri
fugeat4° Candwat erbathat42° C.
PrepareLBbr othbydi ssolv i
ng1. 38gofLBmedi ain55ml ofdistil
l
edwat er
.Steri
l
izeby
autoclaving.
PrepareLBagarpl atesbydi ssolving0.5gofLBmedi aand0. 3gofagari n20mLofst eri
le
di
st i
ll
edwat er.St
eri
li
zebyaut oclavi
ngandpouronst eri
lePetriplate.
Dissolve30mgofampi cil
l
inpowderi n600μLofst eri
ledoubledi stil
l
edwatertogivea
concentrat i
onof50mg/ mL.
PrepareofLBAgarpl atescont ai
ningAmpi cil
li
n,X-
Gal andIPTG( 100ml )byadding100μLof
ampi ci
ll
in,200μLofX- Gal and100μLI PTGt o100mLofaut oclavedLBagarmedi a,mixwelland
pouronst eril
ePetriplates
Day1
1.Opent hevialcontai
ningcul t
ureandr esuspendthepel
letwith0.25mLofLBbrot
h.
2.Pickupal oopfulofcul t
ureandst r
eakont oLBagarplate.
3.Incubateovernightat37° C.
Day2
1.Inocul
ateasi nglecolonyf r
om therevivedplat
ein1ml LBbr oth.
2.Incubateat37° Covernight.
Day3
Take50ml ofLBbr othinast eri
lefl
ask.Transfer1ml ofover
nightgrowncul
tur
eint
othi
sfl
ask.
I
ncubat
eat37° Cshakerat300r pm for3-4hour sti
llt
heO.D600r eaches~0.
6.

PreparationofCompet entCel l
s
Transfertheabov ecult
urei ntoapr e-chill
ed50mLpol ypropyl
enet ube.
All
owt heculturetocool downt o4°Cbyst ori
ngonicefor10mi nut es.
Centri
fugeat5000r pm f
or10mi nutesat4° C.
Decantt hemedium compl etely
.Not racesofmedi um shouldbel ef t.
Resuspendt hecellpell
eti n30mLpr e-chil
l
edsteri
l
e0.1M Cal cium chl ori
desoluti
on.
I
ncubat eonicef or30mi nut es.
Centri
fugeat5000r pm f
or10mi nutesat4° C.
Decantt hecalci
um chlori
desol ut
ioncompl et
ely
.Notracesofsol utionshouldbel ef
t.
Resuspendt hepelleti
n2mLpr e-
chill
edst eri
l
e0.1M Calcium chloridesoluti
on.
Thiscellsuspensioncont ainscompet entcell
sandcanbeusedf ort ransf
ormation.

Transformati
onofcel l
s
1.Take200µLoft heabovecellsuspensi
oni ntwo2.
0mLcollect
iontubesandlabelt
hem as
‘
control’and‘
transf
ormed’.Add2µLofpl asmi dDNAtot
hetubelabeledastr
ansformedand
mixwel l
.
I
ncubat eboththetubesoni cef
or30mi nutes.
Transferthem toapreheatedwaterbathsetatatemper
atur
eof42° Cfor2minut
es(heatshock)
.
Rapidlytransferthet ubest oice-bath.All
owt hecell
stochillf
or5mi nutes.
Add800µLofLBBr ot ht obot ht hetubes.Incubat ethetubesfor1hourat37° Ctoal l
owthe
bacteriator ecoverandt oexpr esst heantibioti
cresist
ancemar kerencodedbyt heplasmid.
Takef ourLBagarpl at escont ainingampi cil
l
in,X-Gal,I
PTGandl abel t
hem ascontrol,
A,BandC.
Plate200µLofcul tur ef rom t he‘control’
tubeandpl at
eitont hecorrespondi
ngplatewitha
steri
lespr eader.Plate50µL, 100µLand200µLofcel lcult
uresfrom the‘t
ransf
ormed’ t
ubeon
tothepl ateslabeledasA, BandC, respectively.
Storeatr oom temper atur et i
l
l t
hepl atesaredr y.
Incubatet heplatesov ernightat37° C.
Afterincubat i
on,obser v
et heplat esforbacter i
algrowthandcountt henumberofv isi
ble
colonies.
I
nterpretati
on
Ontransformationofcell
swithpUC19plasmid,
antibi
oti
cr esi
stancei
sconfer
redont
hehostas
thi
splasmi dcarri
esgeneforampici
ll
inr
esist
ance.Asar esult
,thosecel
l
sthatgrowi
npresence
ofampi cil
l
inaretransf
ormedcell
s.Thetr
ansfor
medcol oniesareblueonX-Gal
,I
PTGplat
esdue
toα-compl ementati
on.