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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.