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BacterialTransformationLab
KaitlinSepanek
MaddieFrew,VictoriaFelder
October52015
Biology2Period2

ObservingBacterialTransformationbyGlowingandGrowingunderU.V.Lighting
withandwithouttheAbsorptionofpGloPlasmidinE.coli

Objective:AfterthetransformationofE.colibytheuptakeofthepGloplasmid,the
bacteriashouldbeabletogrowandhavefluorescence.
IndependentVariable:absorptionofpGloplasmid.
DependentVariable:theabilitytogrowandglowinvaryingenvironmentalconditions.
Controls:pGloLBplate,pGloLBampplate.
Constants:heatshockprocedure,receivingLBbrothafterheatshock,minimumof24
hoursincubationperiodwithincubatorsetat37degreesCelsius(agarfacingdown);
samestrainofE.coli.

Introduction:Inthislab,bacterialtransformationwasused.Thisisaprocessoftakinga
pieceofprotein,orDNA,andinsertingitintoanotherorganismchangingitsgenesand
traits.Genetictransformationisregularlyusedineverydaylife(suchasfoodmaking
processes)andbioremediationforsickpeople(suchaspeoplewithdiabetes).*The

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insulinismadebybacteria,theplasmidintheinsulinattachestotheirDNAand
completesthesequence,healingthemtemporarily.Thislabusestheprocessofbacterial
transformationtomakeE.coli(abacteriumfoundinintestinesofhumansandanimals)
glowbyinsertingthePGLOplasmidfromajellyfish.Heatshockwasusedtocomplete
theprocessoftransformation.Heatshockisdonebyputtingthetesttubesintheicebath,
thenputtingthemintothehotbath,andthenbackintotheicebathagain.Thisprocess
expandsandcontractsthecells,whichmakesthemmorepermeabletoplasmids.A
plasmidisageneticstructurethatreplicateswithoutchromosomes,andisusedto
manipulategenes.Thisplasmid(GreenFlorescenceProteinorGFP)wheninserted
properlywithsugarwillmakethebacteriumglow.Thesugar,arabinose,makesitglow
becausetheprotein,inordertowork,needsanenergysourcetobind.Whenthe
arabinoseispresent,araCbindstothesugarandinstructstheRNAPolymerasetomake
copiesoftheGFPmRNAflorescenceoccurswhenenergyfromanoutsidesourceof
lightisabsorbedbyacellandalmostimmediatelyremittedduetoachemicalreaction
insideanorganism(BioRad).Thisreactionisaidedbyrestrictionenzymes;itcutsboth
endsoftheplasmidneededintheotherDNA,andcreatesstickyendsthatwillattach
easilytoeachother.Oncethestickyendsaretogether,theDNAstrandiscomplete,and
theprocessiscomplete.

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Materials:

250lTransformationsolution

Ice

pGLO

PlasmidDNA

Rack

pGLO

+pGlo

Ice

Incubator

Racksothebottomofthetubesstickoutandmakecontactwiththeice.

4E.coliplates

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Procedure:
TransformationKitQuickGuide
1. Labeloneclosedmicrotesttube+pGLOandanotherpGLO.Labelbothtubes
withyourgroupsname.Placetheminthefoamtuberack.
2. Openthetubesandusingasteriletransferpipet,transfer250loftransformation.
3. Placethetubesoncrushedice.Donotusecubedice.
4. Useasterilelooptopickupasinglecolonyofbacteriafromyourstarterplate.
Pickupthe+pGLOtubeandimmersetheloopintothetransformationsolutionat
thebottomofthetube.Spintheloopbetweenyourindexfingerandthumbuntil
theentirecolonyisdispersedinthetransformationsolution(withnofloating

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chunks).Placethetubebackinthetuberackintheice.Usinganewsterileloop,
repeatforthepGLOtube.
5. ExaminethepGLOplasmidDNAsolutionwiththeUVlamp.Noteyour
observations.ImmerseanewsterileloopintotheplasmidDNAstocktube.
Withdrawaloopful.Thereshouldbeafilmofplasmidsolutionacrossthering.
Thisissimilartoseeingasoapyfilmacrossaringforblowingsoapbubbles.Mix
theloopfulintothecellsuspensionofthe+pGLOtube.Optionally,pipet10lof
pGLOplasmidintothe+pGLOtubeandmix.ClosethepGLOtubeandreturnit
totherackonice.DonotaddplasmidDNAtothepGLOtube.Closethe
pGLGOtubeandreturnittotherackonice.
6. Incubatethetubesonicefor10min.Makesuretopushthetubesalltheway
downintherack.
7. Whilethetubesaresittingonice,labelyourfouragarplatesonthebottom(not
thelid)asshownonthediagram.
8. Heatshock.Usingthefoamrackasaholder,transferboththe(+)pGLOand()
pGLOtubesintothewaterbath,setat42C,forexactly50seconds.Makesureto
pushthetubesallthewaydownintheracksothebottomofthetubesstickout
andmakecontactwiththewarmwater.Whenthe50secondshavepassed,place
bothtubesbackonice.Forthebesttransformationresults,thechangefromthe

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ice(0C)to42Candthenbacktotheicemustberapid.Incubatetubesonicefor
2min.
9. Removetherackcontainingthetubesfromtheiceandplaceonthebenchtop.
Openatubeand,usinganewsterilepipet,add250lofLBnutrientbrothtothe
tubeandrecloseit.Repeatwithanewsterilepipetfortheothertube.Incubatethe
tubesfor10minsatroomtemperature.
10. Gentlyflicktheclosedtubeswithyourfingertomix.Usinganewsterilepipetfor
eachtube,pipet100lfromeachofthetubestothecorrespondingplates,as
shownonthediagramontotheappropriateplates.
11. Useanewsterileloopforeachplate.Spreadthesuspensionsevenlyaroundthe
surfaceoftheagarbyquicklyskatingtheflatsurfaceofanewsterileloopback
andforthacrosstheplatesurface.
12. Stackupyourplatesandtapethemtogether.Putyourgroupnameandclass
periodonthebottomofthestackandplacethestackupsidedowninthe37C
incubatoruntilthenextday.

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DataTable1:Resultsfrombacterialtransformation

Plate
Conditions

Expected
Results

Expected Observed
Results
Results

Observed
Results

PhotoofPlate

Growth

Color
UVand
nonUV

ColorUV
andnonUV

Growth

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Plate1

Yes

UV
yellow
NonUV
yellow

Yes

No

Nocolor

No

Nocolor

Yes

UV
yellow

Yes

UVyellow

LBpGlo
(no
plasmid)
Plate2

UVyellow
NonUV
yellow

LB/amp
pGlo(no
plasmid)
Plate3
LBAmp
+pGlo
(plasmid)

Plate4

LBAmpara
+pGlo

NonUV
offwhite,
yellow

NonUV
yellow

Yes

UV
Green

Yes

NonUV
offwhite

UVGreen

NonUV
yellow,off
white

Conclusion:
Claim:AfterthetransformationofE.colibytheuptakeofthepGloplasmid,thebacteria
shouldbeabletogrowandhavefluorescence.

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Evidence:Indatatableone,theplates1,3,and4grew;however,onlyoneofthose
platesglowedintheUVlight.Inthepicture,itshowstheplatewiththesugarandAmp
geneglowingundertheUVlight.Plate3,theonewithoutthesugar,didnotglowunder
theUVlights,andthecontrolledplate,plate1,didnotglow.Plate2neitherglowednor
grew.
Reasoning:Theplatethatpossessedtheabilitytogrow,Plate4,grewbecausetherewas
sugarprovidedfortheE.colitofeedoffof,andreproducerapidly.Becauseitreproduced
withtheAmp,thepGloplasmidwasputintothenewDNAofE.coli.Theotherplates
mayhavegrownjustasmuch,orevenmorethenPlate4,buttheydidnotglowbecause
oftheabsenceofsugararabinose.Withoutarabinose,thereisnoenergyforthepGlo
genetobeactivated,anddoesnotglowbecauseofthis.Heatshockexpandsandcontracts
thecells,whichmakesthemmorepermeabletoplasmids,whichishowplate4beganto
glow.Plate2didnotgroworglowbecauseitneitherhadthesugarnortheAmpgeneto
reproducelikePlate1,3and4did.Plates1and3grew,becausetheampgenewaspresent
andcombinedtotheDNA,howevertheydidnotglowbecausetheydonothavethe
genestoreproducetheglowinggene,orthefoodtoprovidetheenergytosupportthe
glowinggene.
Reflection: This experiment was properly executed, and nothing was
out of the ordinary in the results obtained from putting the pGlo gene
into the E. coli. The only exception to this would be the pictures of the
controls in the data table. Another group took these photos; however,

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because these controls are all the same, it was acceptable to take the
pictures from them. The experiment, though successful, did not have
some of the same results in other classes. Doing the experiment for
longer time periods while in the ice, hot bath, and incubator, may
increase the percentage of successful results. Doing the experiment
multiple times would also increase the accuracy of each experiment.
The controls, pGloLBplateandpGloLBAmpplate,areintheexperimenttoadd
validity,andcomparisontotheotherplatesthathadthearabinoseand+pGloplasmids
addedtothem.Theconstantscreatedtheisolatedvariablesthatweretestedinthis
experiment;theseareoneofthemostimportantitemsbecauseitdecipherswhatexactly
isbeingtested.Thelevelofconfidenceinthisexperimentwashighbecauseallofthe
controls,constants,andtestedvariableswereintheproperplacesandattherighttimes.

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