Download Enzyme Purification and Plasmid Transformation in E. coli

Enzyme Purification and Plasmid
Transformation in E. coli
By
Scott Bedle
TableofContents:
ListofFigures…………………………………………………..….........1
ExecutiveStatement………………………………………..………….2
Introduction…………………………………………………..………….3-5
Method……………….…………………………………………………….5-6
Results…..………………………………………………………………..7-15
Discussion………………………………………………………………16-17
Conclusion…………………………………………….…...………………17
Citations……………………………………………………………………..18
ListofFigures
Figure 1 – Class Gel electrophoresis……………………………………………….7
Figure 2 – Bacteria grown on +/- amp petri dishes…………………………..…….7
Figure 3 – Single and Double digest of certain Enzymes………………….………8
Figure 4 – APE Xhol Digestion……………………………………….………...…8
Figure 5 – APE Sacl Digestion………………………………………….…………9
Figure 6 – APE EcoRV Digestion………………………………………….……...9
Figure 7 – APE Sacl and Xhol Digested …………………………………….…...10
Figure 8 – APE Sacl and Xhol digested…………………………………….…….10
Figure 9 – APE EcoRV and Xhol Digested………………………….…………...11
Figure10–HypotheticalGel…………………………………………………………………………..….11
Figure11–SDS-Page………………………………………………………………………………………….12
Graph1–Absorbancevs.ProteinConcentration……………………………………………….12
Graph2-DilutionSeries………………………………………………………………………..……………13
Graph3-AveforENZ10^-1……………………………………………………………………………….13
Graph4-.3ONPGsubseries………………………………………………………………………………14
Graph5-.6ONPGsubseries………………………………………………………………………………14
Graph6–AverageofallOPNGvariations………………………………………………………….15
ExecutiveSummary:
EnzymepurificationandingestingitintootherDNAcanrevolutionizemodernmedicine,
farming,andlifeingeneral.Learningabouthowcertainenzymeeffectproteinproductioncan
bedoneusingmanymethods.Thepurposeoftheselabsistoobtainanamountofanenzyme
thatisampicillinresistant,checkitspurityandmakeE.coliingestthisenzyme.Usingcurrent
techniquestodoallofthisE.colicanbemaderesistanttoampicillin.Theenzymeduringthese
labstodothisisBeta-Galactosidase.Usingmodernmethodstheenzymewaspurified,checked
forpurification,andingestedintoE.coli.Theenzymeactivitywasalsolookedatduringthese
labstolookattherelationofconcentrationoftheenzymeandhowiseffectsactivity.
Throughoutthefollowingpagesonewillfindtheinformationabouteachlabinthe
introduction.Inmethodssectiononewillfindabriefdescriptionofthemethodsusedduring
eachlab.Intheresultssectiononewillfindtheresultsfromeachlab.Inthelastsection,
discussionadiscussionoftheresultsandwhattheresultsmeancanbefound.
Introduction:
Theobjectiveoftheselabsistolearnaboutthepreparationandplasmidtransformation
ofE.coli,theexpressionandproductionoftheenzymeBeta-galactosidase,proteinpurification
andanalysisofenzymeactivity.Duringtheseexperimentsandalotofotherresearch
experimentsdealingwithproteins,Escherichiacoli(E.coli)isusedasthebacteria.E.colimakes
goodmodelsformostproteinsandtheoneusedintheselabsinnonpathogenic.Aspecific
strainofDH5-alphaE.coliwasusedduringtheselabs.DH5αisusedforalotofengineering
DNAstrands.ToproduceproteinstheplasmidDNAmustbechanged.Plasmidsaresmall,
circularDNAchromosomeelementsthathaveapartoftheirreplicationcomponent.Plasmids
canbeeasilypurifiedfrombacteriaandtransformedintootherbacteria.Theyalsocanbe
visualizedbygelelectrophoresis.Gelelectrophoresisisamethodforsortingproteins,inthis
case,fortheirsize(Dickey228).TheplasmidusedduringtheselabswastransformedinDH5α
E.coli.Theplasmidusedencodesanampicillin-resistantgene,whichwillshowthepresenceof
theplasmidintheE.coliwhenplacedonpetridisheswithandwithoutampicillin.Acontrolwill
alsobeusedduringtheplatingofE.coli.ToobtainaDNArestrictiondigestmap,theplasmid
mustbedigestedwithaseriesofrestrictionsenzymes.Restrictionenzymesarethescissors
usedbybiologisttocutDNAstrands(Dickey224).TheseenzymescutcertainsectionsofDNA
byneedinganexactstrandofDNAtobindtoit.Usingdifferentrestrictionenzymesanddoing
eitheradoubledigestorasingledigestadigestmapcanbecreated.Tocreatethemapthe
resultedDNAdigestisputintoagarosegel,containaDNAbindingdye,whichwillallow
visualizationoftheDNAinthegel.Themapcanbeusedtocomparesizeandmolecularweight.
TherestrictionenzymesusedduringthemappingoftheplasmidareEcoRV,Xhol,andSacl.A
laddertohelpdetermindmolecularweightandsizewasalsousedtohelpwithGelanalysis.
ThesecanbeusedtomakearelationshipbetweenmovementandsizeontheGel.
Plasmidcanbeobtainedbyextractingitfrombacteria.TodothisaformofAlkaline
lysisisused.Alkalinelysisisamethodofobtainingplasmidoutofbacteriaandcanbeanalyzed
easilybyGelelectrophoresis.ToobtaintheplasmidthepHofthesolutionischangedto
denaturetheDNAandeventuallyaftermanypHchangerenaturetheplasmidwanted
(Brinboim1).AlkalinelysisproducesverypureplasmidDNAthatcanbeusedfor,sequencing,
mutagenesisandrestrictiondigestion.Theplasmidproduced(pET-ELP(11)-lntein(MtuΔCM)lacZ)bythealkalinelysiscanbetestforpurityandcanalsobeingestintootherbacteria.
Theplasmid(pET-ELP(11)-lntein(MtuΔCM)-lacZ)extractedfromtheDH5αE.coliwas
ingestedbyadifferentbacteria,E.coliproteinexpressionstrain(BLR).TheseE.coliwerealso
growninrichmediatoanopticaldensity(OD600)of0.7-0.9,whichmakethecellsgrowfaster
andexponetial(late-logphase).Thebacteriumwasthencooledtohelpwiththecorrectfolding
oftheproteinbeingoverexpressedbytheplasmid.Isopropylβ-D-1-thiogalactopyranoside
(IPTG)wasintroducedtotheE.colitoinducetheproductionofprotein.IPTGisalactose
analogthatreleasestheLacirepressortoactivatethePlac-promotertranscription.PlacpromotercatalyzestheE.colitoexpressthebacteriophageT7RNApolymerase(RNAp),which
isencodedonthebacteria’schromosomes.IPTGisintroducedtothebacteriatoamplifythe
RNApexpressionviathereleaseofLacirepressor.Thisisdoneviaasignalcascade.Asignal
cascadeisachainreactionofsignalingusuallybetweencellstoamplifyproductionofacertain
enzymes(Diwan).IPTGamplifiestheproductionofT7RNApolymerase,whichtranscribesbetaglactosidase.T7RNApisaveryactivepolymerasethatonlyproducesthetargetproteinthatis
encodedontheplasmidwithaT7promoter.SinceIPTGproducedT7RNApandT7RNAponly
producedthedesiredproteinmostoftheproteinproducedbythebacteriumwillbethe
plasmid-encodedprotein.
Inordertopurifythespecificproteinelastin-likepeptidetagging(ELP)reversephase
transition(RPT)musttakeplace.Asampleofcellsthathavebeta-glactosidasefusedwithabifunctionalprotein-purificationtagsequenceengineeredontotheN-terminusisneededto
purifythebeta-glactosidase.ELPtaggingisdonewithtransitionoftemperature.Athigher
temperaturetheELPtaggedproteinfallsoutofthesolution.Aftertheproteinfallsoutofthe
solutionthetagmustbedropped.Inordertodothistheproteinissuspendedbychangingthe
pHto6.0withsalts.Theinteinontheproteinhasbeenengineeredtohelpwiththeprotein
purification.WithanotherroundofRPTtheELPtagcanbedroppedandapureproteinis
formed.TheELPandinteinarebothcontrolledbytemperatureandpHbuttheELPeffectsa
physicaltransformationwhiletheinteineffectstheenzymaticseparation.Samplesduringthe
followingstepscanbetakenandpurificationoftheproteincanbeseenwithSDSPolyacrylamidegelelectrophoresis(PAGE).PAGEusesahydrophilicplasticmatrixmadeof
polyacrylamideinordertoseparateproteinsbasedontheirmolecularweight.Sodiumdodecyl
sulfate(SDS)isusedasthedetergenttounfoldtheproteinsandgivethechainaconstant
charge/massrationforseparation.Afterthegelhasbeenrun(loadingdyeoffrunsoffgel)it
mustbestainedinordertoseethebandsofprotein.CoomassieBlueisthedyeusedtostain
theprotein.
Therearemanywaystotestforproteinpurification.BradfordAssaycanbeusedtotest
theconcentrationoftheprotein.BradfordAssayusesdyetodeterminetheprotein
concentration.CoomassieBrilliantBlueG-250thedyeusedinthisexperiment.Inorderto
formarelationshipbetweentheabsorbanceandproteinconcentrationagraphmustbemade.
Thestandardcurveusesknowconcentrationofaknowprotein,BovineSerumAlbumin,inorer
tomakethisrelationshipforbeta-galactosidaseandthedyeused.Adirectspectrophotometric
measurementofproteincanbetakenusingananodropspectrophotometer.Beerslawmust
thenbeusedtocalculatetheconcentration.Proteinshaveanaverageabsorbancepeakat
around280nmandthismustbeusedwiththenanodropspectrophotometer.Nanodrop
spectrophotometercanastestforhowpuretheproteinisbycomparingittotheabsorbanceof
DNA.Totestthis,theproteinandDNAaretestatboth260nmand280nm.These
absorbancesaregraphedandthepercentagedifferencebetweenthetwodetermineshow
puretheproteinis.
Enzymeactivitycanbetestusingthespectrophotometer.O-nitrophenylbeta-D-
galactoside(ONPG)canbeusedtotestactivityofbeta-galactoseinasolution.ONPGturnsinto
glactoseandayellowo-nitrophenol.Theyellowo-nitrophenoliswhatthespectrometerpicks
uponduringthereactioncausinganincreasedabsorbancereading.Thisincreasein
absorbancereadingcanmeasuretheactivityofenzymes.Differentconcentrationofboth
ONPGandtheenzymecanbetestedtofindthebestreactionconcentrationsforanenzyme.
Methods:
Gel Electrophoresis:
A plasmid of DNA sample is loaded into the Gel and electricity is run through the Gel
allowing the DNA because it is negative to run down the Gel. A UV light camera can take a
picture of the Gel (Dickey 229)
Modified Alkaline Lysis:
The modified Alkaline Lysis that is used is done by changes of temperature and pH to
purify the protein. This makes the ELP tagged protein suspend and resuspend. The intein then
drops off the protein leaving just the Beta-Gal. During each step the solute becomes more pure
than the last until just the Beat-Gal remains.
PAGE:
To run the digests each one was loaded into a gel with a buffer and an electric current
was run through them. After the gel has been completely run the Gel is heated and the dye is
added. After the dye is heated in a microwave the Gel is then clean by heating it in water to get
excess dye out. A normal photograph can be taken of this gel.
Mini Prep:
The Qiagen Mini Prep was used during this experiment. This is used to extract a
bacterial plasmid DNA. First the cells must be lysed to denature then neutralized to renature
DNA but denatures and helps unfold the protein. Potassium-SDS must be used to precipitate
DNA and some protein but not plasmid DNA. Once the plasmid DNA is caught in a spin
column it is washed and then removed from the column as a pure plasmid DNA.
Transforming Bacteria:
To get bacteria to take in a selected plasmid DNA the cells must be heat shocked. This
will cause the cells to ingest the plasmid DNA. The mini prep above can produce the plasmid
for the bacteria to ingest.
Nanodrop Spectrophotometer:
The nanodrop spectrophotometer needs a small amount of protein to test the
concentration of the protein and purity. It does this by using Beer law and graphing the results.
APE Software:
The APE software used has an coded plasmids (Beta-Gal) and allows one to added
different enzyme to cut the plasmid and shows when the plasmid will be cut and can graph it to
show what it would look like when run on a gel.
Absorbance Spectrometer:
The machines tests for the absorbance of during enzyme reactivity and the date from this
can be plotted to find the concentration of the protein being tested. The machine picks up on the
yellow color produced during the reactivity of the enzyme and ONPG.
Procedure for all methods:
The procedure of all of these methods can be found either online, in the sources at the end
of the report or the lab protocol given.
Results:
Figure1–ClassGelelectrophoresis
ThefigureaboveshowstherunfortheuncutplasmidDNAfortheentireclass.TheladderDNA
sampleisonthesidetocomparewiththeuncutplasmidDNA
Figure2–Bacteriagrownon+/-amppetridishes
ThefigureaboveshowsE.Coliwiththeplasmidinitandwithouttheplasmidinit.Theywere
bothputonpetridisheswithampicillinandwithoutampicillin.
Figure3–SingleandDoubledigestofcertainEnzymes
TheabovefigureshowsplasmidDNAwithcutsfromcertainenzymes.Theupperoneisthe
samplebeinglookedatforthisexperiment.
10545 XhoI (2)
F1 ori 457..17
M13 origin 12..467
AmpR 797..1456
B-Gal 7450..10529
ColE1 origin 1554..2236
pET-ELP11-Intein-Beta-gal_annotated map
10707 bp
7396 XhoI (2)
DI-CM Intein 6946..7449
lacI(q) 4730..3648
ELP 5209..6873
LacO 5136..5158
Figure4–APEXholDigestion
TheabovefigureshowsthedigestionofXholenzymeintheBeta-GalplasmidonAPEsoftware.
F1 ori 457..17
M13 origin 12..467
AmpR 797..1456
9400 SacI (2)
B-Gal 7450..10529
ColE1 origin 1554..2236
pET-ELP11-Intein-Beta-gal_annotated map
10707 bp
DI-CM Intein 6946..7449
6879 SacI (2)
lacI(q) 4730..3648
ELP 5209..6873
LacO 5136..5158
Figure5–APESaclDigestion
TheabovefigureshowsSacldigestedintotheplasmidonAPEsoftware
F1 ori 457..17
M13 origin 12..467
AmpR 797..1456
B-Gal 7450..10529
ColE1 origin 1554..2236
8575 EcoRV (2)
pET-ELP11-Intein-Beta-gal_annotated map
10707 bp
DI-CM Intein 6946..7449
ELP 5209..6873
3927 EcoRV (2)
lacI(q) 4730..3648
LacO 5136..5158
Figure6–APEEcoRVDigestion
TheabovefigureshowsEcoRVdigestedintheBeta-GalplasmidonAPEsoftware.
10545 XhoI (2)
F1 ori 457..17
M13 origin 12..467
AmpR 797..1456
9400 SacI (2)
B-Gal 7450..10529
ColE1 origin 1554..2236
pET-ELP11-Intein-Beta-gal_annotated map
10707 bp
7396 XhoI (2)
DI-CM Intein 6946..7449
6879 SacI (2)
lacI(q) 4730..3648
ELP 5209..6873
LacO 5136..5158
Figure7–APESaclandXholDigested
ThefigureaboveshowsthedoubledigestofbothSaclandXholintheBeta-Galplasmidonthe
APEsoftware.
F1 ori 457..17
M13 origin 12..467
AmpR 797..1456
9400 SacI (2)
B-Gal 7450..10529
ColE1 origin 1554..2236
8575 EcoRV (2)
pET-ELP11-Intein-Beta-gal_annotated map
10707 bp
DI-CM Intein 6946..7449
6879 SacI (2)
ELP 5209..6873
3927 EcoRV (2)
lacI(q) 4730..3648
LacO 5136..5158
Figure8–APESaclandXholdigested
ThefigureaboveshowsthedoubledigestofbothSaclandXholdigestedintheBeta-Gal
plasmidontheAPESoftware
10545 XhoI (2)
F1 ori 457..17
M13 origin 12..467
AmpR 797..1456
B-Gal 7450..10529
ColE1 origin 1554..2236
8575 EcoRV (2)
pET-ELP11-Intein-Beta-gal_annotated map
10707 bp
7396 XhoI (2)
DI-CM Intein 6946..7449
ELP 5209..6873
3927 EcoRV (2)
lacI(q) 4730..3648
LacO 5136..5158
Figure9–APEEcoRVandXholDigested
ThefigureaboveshowsthedoubledigestofEcoRVandXholdigestedintheBeta-galplasmid
ontheAPEsoftware.
Figure10–HypotheticalGel
Figure10showswhatFigure3shouldlooklike.
Figure11–SDS-Page
Theorderofthesamplesrungoeswholelysate,clarifiedlysate,solublecontaminates,purified
precursor,post-cleavingproteins,purifiedprotein,precipitatedtag,beta-gal,apurifiedprotein,
aladder,andacontrol.
Absorbancevs.Protein
Concentration
Absorbance
25
20
15
10
y=87.926x+1.8751
5
0
-0.025 0
0.025 0.05 0.075 0.1 0.125 0.15 0.175 0.2 0.225
Concentration(ug/ml)
Graph1–Absorbancevs.ProteinConcentration
ThegraphaboveshowstheAbsorbancevs.theconcentration.
DilutionSeries
0.45
0.4
0.35
Absorbance
0.3
0.25
ENZ10^-1
0.2
ENZ10^-2
0.15
ENZ10^-3
0.1
0.05
0
-0.05
0
50
100
150
200
250
300
Time(sec)
Graph 2- Dilution Series
The graph above shows the enzyme that was least diluted had the most activity while the other
two dilutions did not reacts as much.
aveENZ10^-1
0.45
0.4
Absorbance
0.35
0.3
0.25
0.2
aveENZ10^-1
0.15
0.1
0.05
0
0
50
100
150
200
250
Time(Sec)
Graph 3 - Ave for ENZ 10^-1
300
The graph above shows the average absorbance over a 4-minute period of the fastest dilution
series (ENZ10^-1).
.3ONPG
0.45
0.4
Absorbance
0.35
0.3
0.25
Series1
0.2
Series2
0.15
Series3
0.1
0.05
0
-0.05 0
50
100
150
Time(sec)
200
250
300
Graph 4 - .3 ONPG sub series
This graph shows the activity of the enzyme in a solution with .3 ONPG.
.6ONPG
0.5
Absorbance
0.4
0.3
Series1
0.2
Series2
Series3
0.1
0
0
-0.1
50
100
150
200
250
Time(sec)
Graph 5 - .6 ONPG sub series
300
The graph above shows the activity of .6 ONPG in a solution with the enzyme.
0.45
SubstrateSeries
0.4
0.35
0.3
ave.1
Absorbance
0.25
ave.2
0.2
ave.3
0.15
ave.4
ave.5
0.1
ave.6
0.05
0
0
-0.05
50
100
150
200
250
300
Time(sec)
Graph 6 – Average of all OPNG variations
The graph above shows the activity of a standardized enzyme mix with solution set a 2.7ml but a
different amount of ONPG each time.
Results for Nanodrop Spectrophotometer:
The A280 for the Beta-Gal is .045 and the 260/280 ratios is .33 meaning that our Beta-Gal is not
very concentrated but extremely pure.
Discussion:
TheuncutplasmidofDNAshowswheretheproteiniscomparedtotheladder.Using
Figure10onecanseewhichenzymecanbeusedandwhentheyshouldbeusedtocutoutthe
Beta-Galprotein.
Theresultsforourcoloniesofcellswereasexpected.Thecoloniesinplasmidhadthe
abilitytosurviveintheampicillinwhereasthecellswithouttheplasmidcouldnotsurvive.Both
cellssurvivedandthrivedinthegelwithoutampicillin.
OurcelldigestsorderwasLadder,Control,Sacl,Xhol,EcoRv,EcoRvandXhol,Sacland
Xhol,EcoRvandSacl.Clearlysomethingwentwronginthedigestofourenzymes.Acouple
factorsthatcanbebroughtintothefailedexperimentcouldbe.Theamountofenzymethat
madeitintothedigestwasnotpresent.Thisisbecausethepipetisonlymeasurableat2.0
microlitersandwehadtoput.5microlitersin.Ifoneweretocometoaconclusiononthe
relationshipbetweenmassandmigrationlookatanAPEmapofthedigestthemoreenzymes
thatcuttheDNAthefartheritmoves.Sothesingledigestsonlymovesofarbecausetheyhave
alotofmasswhilethedoubledigestsmoveabitmorebecausetheyhavelessmass.
Onecanseethatastheprocessofpurificationgoesonmoreandmorejunkgetstaken
outandevenwithalltheproblemsencounteredduringtheselastcouplelabs,forinstancethe
solutenotre-suspendingmultipletimes,theproteinwasstillabletobepurified.Bylookatthe
gelitconfirmsthatthesampleasapurifiedproteininthebeta-galsampleandalsothepurified
proteinsample.Thisproteinthatwasconfirmedwillbeusedinlaterlabs.
Usingthestandardcurvegraphaboveandpluggingintheabsorbanceoftheunknown
concentration(-.008)thestandardequationgivestheunknownaconcentrationof1.18
micrograms/ml.ThisfitswiththeSDS-Pagegel,whichconfirmedtheproteinwaspresentand
purified.Aproblemranintoduringthisprocesswasnegativeabsorbancenumbersforsamples
thathadaconcentration.Thisflawedourstandardcurvebecausethevalueswereoff.Abetter
waytofindtheequationwouldbetoincreasetheknowconcentrationsotheyarenotassmall.
Muchcanbelearnedfromboththedilutionseriesandthesubstrateseries.Duringthe
dilutionseriesitcanbeseenthatthemoredilutedtheenzymetheslowerthereaction.The
highestconcentrationoftheenzymereactsmuchfasterthanthe2ndor3rddilutions.Lookingat
thesubstrateseriesonecancometotheconclusionthatthelowerdilutionsstartatfastbut
slowdownwhereasthehigherconcentrationsstartfastandcontinuetostayatafasterrate.
The.6solutionofONPGalthoughstayverysimilartothe.3dilutionseries.
Problems:
Acoupleofproblemshappenedduringthisexperiment.Thefirstproblemwasthatthe
bacteriathatwassupposetobeuseddidnotreproducesoDr.Lease’sE.Colihadtobeused.
Theotherproblemencounterwassomethinghappenwiththedigestthatmadethemnotrun
liketheyshouldhave.Fortunately,agelusingAPEwascreatedtomimicwhatisshouldhave
lookedlike.Thisgelshowstherelationshipbetweenmassandhowfarthegelplasmidwillrun.
UpdatestoLabprotocol:
Althoughthelabprotocolwasveryhelpfulandinformativeitcouldusesome
improvement.Oneimprovementtotheprotocolcouldbethattheprocedurebewrittenmore
clearlysoitcanbeunderstoodbynon-biologypeoplewhowanttopreformthisexperiment.
TheprocedurewasverythickinwashardtofollowwithoutRyanBell’shelponexplainingwhat
todo.Alsoalittlemorebackgroundinformationshouldhavebeengivenintheprotocolitself
sonotasmuchoutsidereadingandresearchisneededtounderstandkeyaspectsofthe
protocol.Theseupdateswouldonlyhelpstudentunderstandthelabbetterandwouldallowa
non-biologystudentcompletethelabwithoutandaninstructor’shelp.
Conclusion:
EnzymepurificationandingestingitintootherDNAcanrevolutionizemodernmedicine,
farming,andlifeingeneral.Learningabouthowcertainenzymeeffectproteinproductioncan
bedoneusingmanymethods.Theobjectiveoftheselabswastolearnaboutthepreparation
andplasmidtransformationofE.coli,howtoexpressandproducetheenzymeBetagalactosidase,howtopurifyproteinbytheuseofpurificationmethodsandtheanalysesof
enzymeactivity.Throughalltheselabsonecanlearnhowtoproduceproteinsandenzymes
andhowenzymescatalyzereactions.
Citation:
Banki,Mahmoud,LiangFeng,andDavidWood."Simplebioseparationsusingself-cleaving
elastin-likepolypeptidetags."(2005):n.page.Print.
Birnboim,H.C.,andJDoly."A
rapidalkalineextractionprocedureforscreeningrecombinantplasmidDNA."Nucleic
Research.France:1975.
Diwan,Joyce.SignalTransductionCascades.Diss.RensselaerPolytechnicInstitute,2008.Print.
<http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/signals.htm>.
Simon,Eric,JeanDickey,andJaneReece.EssentialBiologywithPhysiology.Fourth.London:
DorlingKinderleyLimited,2013.Print.