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Introduction:
GeneticengineeringinvolvesmodifyinganorganismsDNAtodeliberatelychangeanaspectofthe
organismforaparticularpurpose.ThiskitdemonstratesthepoweroftheCRISPRCas9systemby
modifyingthegenomicDNAofastrainofE.colisothatitcangrowandsurviveinconditionsit
normallywouldnotbeableto.

Thiskitrequires~10hoursofworkoverthecourseofatleast2days.Itcanbecompletedina
weekendiffreshbacterialculturesarepreparedonaF ridaynight.

Asthisdocumentisconstantlybeingupdatedwithtipsandpointersandtherearevideolinks
embedded,youcanfindthemostuptodateversiononlineat:h ttp://goo.gl/YBfYuQ

WhatisCRISPRCas9doinginthisexperiment?
Bacteriaandallorganismsneedtomakeproteinstosurvive.Proteinsaretinynanomachinesthatdo
everythingfromcontrolourmetabolismtokeepingourheartbeating.Inordertomakeaproteinacell
usestheDNAcode.Each3lettersofDNAcodesforasingleaminoacidandproteinsarejustchains
ofaminoacids.

Proteins(likecas9)aremadebyanucleicacidandproteincomplexinthecellcalledther ibosome.
Themediathatyouareattemptingtogrowthebacteriaoncontainsamoleculecalledstreptomycin
whichbindstheribosomeandpreventsitfrommakingproteins,notallowingthebacteriatoreplicate
andsotheycantgrow.Thiskitmakesaspecificmutationintheribosomalsubunitproteinr psLthat
preventsstreptomycinfrombindingitandsothebacteriacangrowjustfineonthemedia.

ThegenomeoftheE .colibacteriaisthatyouwillengineerisover4millionDNAbasesinsizeand
CRISPRwillfindthesingleonethatneedstobemutated!Thismutationwillcauseanaminoacid
changeintheribosomalsubunitproteinsthatarebeingmade.

FormoreinformationonsequencesanddetailscheckoutourmoreadvancedCRISPRguidehere

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Kitcontents(p g.3)

Timeline(p g.4/5)

MakingPlates(p g.6/7/8)

MakingCompetentBacteria(p g.9/10/11)

DNATransformation&CRISPR(p g.12/13)

Successfulexperimentexample(p g.14)

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1LBAgar(6gina15mLtube)

1LBStrep/KanAgar(6gina15mLtube)

1250mLglassbottleforpouringplates(fillwith100150mLwater)

110100uLvariablevolumeadjustablepipette(1uLincrements)

1Box(96count)1200uLPipetteTips

14PetriPlates

1Microcentrifugetuberack

10InoculationLoops/Platespreaders/PairsNitrileGlovesinplasticbag

50microcentrifugetubes

51.5mLmicrofugetubescontaining.03gLB

50mLcentrifugetube

1mLbacterialtransformationbuffer25mMCaCl2,10%PEG80005%DMSO

Perishables
E.coliHME63strain

Cas9andtracrRNAplasmid55uLof100ng/uL

crRNAplasmid55uLof100ng/uL

TemplateDNA55uLof100ng/uL

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Preparation
1hourMakeplates(setasidemoretimeifit'syourfirsttimemakingplates)
streakoutbacteriaontoanLBAgarplate(takes~1min)
1218hoursL etthebacteriagrow(easiesttojustletitsitovernight)

Dayofexperiment
Mixtogethersample,plasmids,andtransformationmix(takes~5min)
30minrefrigeratesamplesolution(doNOTfreeze)
30secondsheatshockthesamplewarm(42C/108F)water.AddLBmediatoyourcell
solution(takes~1min)1hourincubatefor1hour,(orif@roomtemp,incubatefor3hour)1
0
minPlate100uLofthebacteriasolutionandletdryfor10minutes

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Incubateandwaitforgrowth

~24hoursIncubatetheplateat37C(99F)for1624hoursorr oomtemperaturefor2448
hours.

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MakingPlates(~1hour,maybemoretimeifitsyourfirsttime)
Stepbystepwalkthroughwithphotosat:h ttps://goo.gl/7yzpA1

Agarplatesprovideasolidmedianutrientsourceforbacteriaandyeasttogrowon.Thestandard
mediathatisusedisLB(LuriaBroth,LysogenyBroth,orLuriaBertaniBroth).Thiscontainsacarbon
source,anitrogensource,andsalt(manystrainsofbacterialikesalt!).

Thetoppartofthefullplatehasthelargerdiameter.

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MakingPlates
1. TakeatubelabelledAgarmedia,suchasLBAgarMedia,LBStrep/KanAgarMedia(For
finalgrowthtest)orsimilaranddumpitscontentsintothe250mLglassbottle.(Youwillneedto
makeplatesoutofeachkindofmedia,sostartwithwhichevertubeofmediayouchoose.)
2. Usingthe50mLconicaltubelabelledForMeasuringWater,measureandadd150mLof
watertotheglassbottle.
3. Makingagarislikemakingjelloheattheagartodissolveit,thenitwillsolidifywhenitcools.
Heatthebottleinthemicrowavefor30secondsatatime,beingcarefulnottoletthebottleboil
over.DONOTSCREWTHELIDDOWNTIGHT!(justplaceitontopandgiveitaslightturn)
4. Youwillknowitsdonewhentheliquidlooksyellowandfullyseethrough(nofogginess).This
shouldtakeabout23minutestotalofmicrowaving.Takethebottleout(cautioncontentshot)
andletitcooluntilyouareabletotouchitwithoutmuchdiscomfort.Thiswilltake2030
minutes.
5. Whilethebottleremainssomewhatwarm,pourtheplates.Oneatatime,removethelidof7
platesandpourjustenoughoftheLBagarfromthebottletocoverthebottomhalfoftheplate.
Putthelidbackon.

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MakingPlates
6. Letcoolforatleastone1hourbeforeuse(youcancoolfasterbyputtingtheminthefridgebut
dontfreeze).Ifpossiblelettheplatessitoutforacouplehoursorovernighttoletthe
condensationevaporate.Thenstoreinyourfridgeat4Cupsidedownsoanycondensation
doesntdripontheplates.

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MakingCompetentBacterialCellsforTransformation
CompetentmeansthebacteriaoryeastcellsareabletointakeforeignDNA.Thecellswalls
normallypreventthingsfromenteringin,butwearegoingtomixthebacteriawithchemicalsand
saltsthatchangethis.InordertogetCRISPRtoworkinsidethebacterialcellsweneedtogetallof
thecomponentsinsidethecells!Thisprocessiscalledtransformation.Weputallthematerialsinto
syntheticDNAandthentrickthebacteriaintothinkingthatourDNAisitsownDNAandsotheymake
theCas9protein,thetracrRNAandcrRNA.

Thebacterialtransformationmixcontains:
10%PolyethyleneGlycol(PEG)3350
PEG3350isthoughttoplayseveraldifferentrolesintransformation,thoughnobodyreallyknowsfor
certain.SincebothDNAandcellwallsarenegativelycharged,theyrejecteachother.PEG3350is
thoughttofunctionbyshieldingthechargeoftheDNA,therebymakingiteasiertopermeatethecell
wall.PEG3350isalsothoughttohelptransporttheDNAintothecell,aswellasmakethecell
membraneitselfmoreporous.

Thispaperfrom1989testsoutdifferenttransformationmixesanddeterminesthatPEG3350works
bestforE
.coli.Thatsonpage2173.
http://www.pnas.org/content/86/7/2172.long

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MakingCompetentBacterialCellsforTransformation
5%DimethylSulfoxide(DMSO)
DMSOissometimesusedtotreatailmentsinhumans.Inatransformationitisthoughtto
permeabilizethecellwall.Also,sometimesDNAfoldsintocomplexstructuresthatmakeitmore
difficulttopassthroughthecellwall.DMSOalsomighthelptobreakthesestructuresdown.

25mMCalciumChloride(CaCl2)
SimilarlytoPEG3350,CaCl2isthoughttoshieldandneutralizethenegativechargeofDNA,thereby
makingitmorelikelytoenterintothecell.

1. Streakoutanewplateofbacteriausinganinoculationloopandletitgrowovernight~1218
hours,oruntilyouseewhiteishbacteriabegintogrow.UsetheLBagarplate,N
OTthe
LB/Strep/Kanagarplate.Seethefollowinglinkforawalkthroughofhowtostreakout
bacteria:h ttps://goo.gl/GR8IOf
a. Note:avoidplacingtheplateinareasofhightemperaturevariationlikeanunheated
garage.Consistentandwarmtemp.locationsarepreferable.
b. Havingfreshbacteriaforatransformationgreatlyincreasesthelikelihoodthatyour
experimentwillwork.

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MakingCompetentCellsforTransformation
2. Pipette100uLofTransformationmixtoanewmicrocentrifugetube.Seethefollowinglinkto
learntopipette:h
ttps://goo.gl/nrA8hT

3. Usinganinoculationloop,gentlyscrapesomebacteriaoffofyourfreshplateandmixitintothe
transformationmix.Mixuntilanybigclumpshavedisappeared.Thismightrequiregently
pipettingthemixtureupanddown.Avoidcreatingbubblesifpossible.Yourtransformationmix
shouldbeverycloudy,ifnotmixinmorebacteriatillyoucannoteasilyseethroughtheliquidin
thetube.MakeonetubeforeachCRISPRexperimentyouplantoperforminthenextdayor
twoandstorethemat4C(39F)inthefridge.

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WatchthisVideoaboutCRISPRitwillhelpyouunderstandhowitworks:
https://www.youtube.com/watch?v=2pp17E4EO8

CRISPRHas3Mainparts

Cas9Protein
TheCas9proteinistheengineofCRISPR.ItbindsthegRNAandalsothegenetargetedforediting.
Ifagenematchisfound,theCas9proteinwillcutthetheDNA.Thecellrespondstothecutbytrying
torepairtheDNAdamage.Cas9cuts,itDOESNOTdoanyactualgeneediting.Instead,ittricksthe
cellintodoingit.

guideRNA(gRNA)
ThegRNA,isarecentinvention.ItisactuallyacombinationofthetransassociatedCRISPRRNA
(tracrRNA)andtheCRISPRRNA(crRNA),connectedbyasmallnucleotidelinker.Somepeopleuse
theseparatetracrRNAandcrRNAforimprovedefficiencies.IntheDIYBacterialCRISPRkitwewill
useseparatetracrRNAandcrRNAs.ThetracrRNAbindstotheCas9proteinandthecrRNA.The
crRNAbindstothetracrRNAinordertoconnectedtotheCas9protein.Critically,thecrRNAmatches
(iscomplementaryto)theDNAinthegenomethatwewanttoedit.ThiscrRNAmatchishowtoCas9
proteinrecognizesthegenetocut.

TemplateDNA
OncetheCas9proteinmakesacutonthegenewewanttoedit,thecellbeginstotryandrepairthe
DNAthroughaprocesscalledHomologousRecombination.Duringthisrepairprocess,thecellis
lookingforaDNAtemplatetofigureouthowtofillinthegenethatwascut.Ifwefloodthecellwitha
templatethatissimilartothemissingregion,buthasamutationorchangeinit,thecellwillmistakeit
foratruecopyanduseitinstead.OurtemplateDNAhasasinglebasechangefromanAdenine(A)
toaCytosine(C).ThischangecausestheDNAtocodeforaLysineinsteadofaThreonineinan
importantprotein.ThischangepreventsStreptomycinfrombindingtoanddisablingtheprotein,which
allowsthebacteriacelltogrowonmediacontainingit.

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1. FindtheDNAtubelabelledCas9andtracrRNAand,usingyourpipette,add10uLtoyour
competentcellmixture.Changeoutthepipettetipforanewone.
2. FindtheDNAtubelabelledcrRNAand,usingyourpipette,add10uLtothesamecompetent
cellmixturethatyouaddedtheCas9andtracrRNAto.Changepipettetips.
3. FindtheDNAtubelabelledTemplateDNAand,usingyourpipette,add10uLtothesame
competentcellmixturethatyouaddedtheCas9andtracrRNA,andcrRNAto.
4. Incubatethistubeinthefridge(DONOTFREEZE)for30minutes.
5. Incubatethetubefor30secondsin42C(108F)water.Youcanapproximatethistemperature
byusingwaterthatiswarm,butcomfortableenoughsuchthatyoucanstillkeepyouhandinit.
6. Add1mLofroomtemperaturewatertooneoftheLBmediamicrocentrifugetubesandshake
todissolvetheLB.
7. Usingthepipette,add200uLofLBmediatoyourcompetentcellmixturecontainingyourDNA.
8. Incubatethetubeat37C(99F)for2houror4hoursatroomtemperature.Thisstepallowsto
bacteriatorecoverandreplicatetheDNAandperformtheCRISPRengineeringprocess
_DONT_Skimponthetime,thisstepiskeyfortheexperimenttowork.TakeaLB/Strep/Kan
plateoutofthefridgeandletitwarmuptoroomtemperature.
9. Usingthepipette,add100uLofyourCRISPRtransformationmixtureontopofanLB
Strep/KanAgarplate.
10. Usinganinoculationloop,gentlyspreadthebacteriaaroundtheplateandletdryfor10
minutesbeforeputtingthelidbackon.
11. Fliptheplateupsidedowntopreventcondensationfromforminganddrippingontoyour
bacteria.
12. Incubatetheplateat37C(99F)for1624hoursorroomtemperaturefor2448hours.

13. Ifyoubegintoseelittlewhiterounddotsgrowing,thenyourCRISPRgenomeengineering
experimentwasasuccess!Ifnot,giveitanothershot,Sciencedoesntalwaysworkonthefirst
try.Also,feelfreetocontactusatodin@theodin.comandwewillhelpyoutroubleshoot.

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Successfulexperimentexample...



Inasuccessfulexperimentyoushouldseewhitishoryellowishbacteriagrowingontheplateasseen
inthepicture.Thesearebacterialcoloniesthatweresuccessfullyeditedandsotheysurvivedand
replicatetoformwhatScientistscallcolonies,orsmallgroupsofbacteria.

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