LastUpdate:April,2020
Grade:Grade11/12 Subject:Biology Unit:Genetics/MolecularBiology
Title:SeparationExploration–TheScienceoftheSeparationofMixtures
RationaleMixturesfrequentlyneedtobeseparatedinbiology,biochemistryandchemistry.Thescientistmightneedtoseparateonetypeofantibodyfrommany,separatepiecesofDNAfromanotherbysize&charge,orneedtopurifyaproteinforlateruse.AlloftheselaboratorytechniquescomefromtheconcepttaughtinGrade9Scienceabouttheseparationofmixturesbasedonphysicaland/orchemicalproperties.Later,inGrade11and/orGrade12Biology,theconceptcomesupagainingelelectrophoresis.Theseconceptsarere-visitedandexpandeduponinuniversitychemistrycourses,suchasanalyticalchemistry.Theseanalyticaltechniquesarewidelyusedinindustry.
BackgroundInformationTheWakarchuklabatRyersonUniversity(undertheumbrellaofGlycoNet)isacarbohydrateresearchlabthatusesenzymesfortherapeuticpurposes.Someofthesepurposesare:
• Theuseofenzymestoaddsugarstoproteindrugstomakethesedrugslastlonger(persist)inthebloodstream,therebyreducingdosage,sideeffectsandcost
• Addingsugarmoleculestoculturedneuronalcellsurfacesusingenzymes,sothatthesemodifiedneuronswillfindtheirwaytowheretheyareneededwhenplacedinParkinson’spatients
• Researchinghowtomaketheseproteinsusingbacteria(ratherthanmaintainexpensivemammaliancelllines)
Inthislaboratory,therearemanytechniquesthatareusedtoseparatemixturesand/oranalyzethem.Thesetechniquesinclude:
• FPLC(fastproteinliquidchromatography)/HPLC(highperformanceliquidchromatography)
• GelelectrophoresisandSDS-Page• TLC(ThinLayerChromatography)
ForfurtherinformationabouttheresearchdoneattheWakarchuklab,visit:https://www.wakarchuklab.com/
LastUpdate:April,2020
CurriculumConnections:Grade11Biology:GeneticProcesses
• D1.1analyse,onthebasisofresearch,someofthesocialandethicalimplicationsofresearchingeneticsandgenomics(e.g.,geneticscreening,genetherapy,invitrofertilization)[IP,PR,AI,C]
• D1.2evaluate,onthebasisofresearch,theimportanceofsomerecentcontributionstoknowledge,techniques,andtechnologiesrelatedtogeneticprocesses(e.g.,researchintothecysticfibrosisgene;theuseofsafflowerstoproduceinsulinforhumanuse)[IP,PR,AI,C]
• D3.4describesomegeneticdisorderscausedbychromosomalabnormalities(e.g.,non-disjunctionofchromosomesduringmeiosis)orothergeneticmutationsintermsofchromosomesaffected,physicaleffects,andtreatments
• D3.5describesomereproductivetechnologies(e.g.,cloning,artificialinsemination,invitrofertilization,recombinantDNA),andexplainhowtheirusecanincreasethegeneticdiversityofaspecies(e.g.,farmanimals,crops)
Grade12UBiology–Biochemistry
• B1.1analysetechnologicalapplicationsrelatedtoenzymeactivityinthefoodandpharmaceuticalindustries(e.g.,theproductionofdairyproducts;breadmaking;theuseofenzymestocontrolreactionratesinpharmaceuticals)[AI,C]
• B2.1useappropriateterminologyrelatedtobiochemistry,including,butnotlimitedto:activeandpassivetransport,covalentandionicbond,allostericsite,substrate,substrate-enzymecomplex,andinhibition[C]
Grade12UBiology–MolecularBiology
• D2.3conductaninvestigationtoextractDNAfromaspecimenofplantoranimalprotein[PR]
• D3.4explainhowmutagens,suchasradiationandchemicals,cancausemutationsbychangingthegeneticmaterialincells(e.g.,themechanismsandeffectsofpointmutationsandframeshiftmutations)
• D3.5describesomeexamplesofgeneticmodification,andexplainhowitisappliedinindustryandagriculture(e.g.,theprocessesinvolvedincloning,orinthesequencingofDNAbases;theprocessesinvolvedinthemanipulationofgeneticmaterialandproteinsynthesis;thedevelopmentandmechanismsofthepolymerizationchainreaction)
LastUpdate:April,2020
LessonObjectives/Concepts
• StudentswilllearnaboutanalyticaltechniquesusedinindustryinaCanadianresearchlaboratorylocatedinToronto,ON(RyersonUniversity)bylisteningtovideoclipsfromCanadianresearchersdiscussingtheircurrentcarbohydrateresearch
• Grade11/12UBiologystudentscanchoosetodoaDNAExtractionInquiry
TimePeriod1:30minutesinthecomputerlab&30minreadingchosenarticlePeriod2:DNAExtractioninquiry:fullperiod
Materials:Period1:Videoclips&Literacyconnection
• Studentsshouldbringtheirownearphonesforuseinthecomputerlab(orlistentothevideosathome)
• Computerfiles:o SeparationExploration-Gr.11/12o InkChromatographylabreport&rubric
Period2:MaterialsforDNAExtractionInquiry
• SoftFruit(mango,bananas,kiwis,dragonfruit,avocado,etc)• Filters(cheesecloth,filterpaper,mesh,stockings,filterpaper,etc)• Chilledrubbingalcohol–about10mLpergroup(4degreesCelsius,fridgetemp)• 2Beakers• Elasticband• Largetesttube(25ml)• Mortarandpestle(1pergroup)orthefruitcanbemashedinsideaZiplocbag• Saltwater(1tspdissolvedin0.5cupofwater)• Detergent(1tspper1banana)• Woodensplintorwoodencoffeestirstick
OverviewPeriod1(libraryperiod)PartA:ListentotheScientists
• Studentsuse“SeparationExploration-Gr.11&12”toguidethemthroughthevideo
LastUpdate:April,2020
clipsoftheGlycoNetresearchers.PartB:LiteracyConnection
• ReadtheJune9,2017CNNarticle“TheShiftingScienceofDNAintheCourtroom”byChannonHodge:http://www.cnn.com/2017/06/09/health/dna-technology-forensic-evidence/index.html
• StudentscompleteachartthathasthemlookingOntheLines(5WsandHow),BetweentheLines(inferencing)andBeyondtheLines(consequences).
Period2:Grade11&12Biology:DNAExtractionInquiry(fullperiod)StudentsbegintheperiodbyreadingtheexperimentalprocedurefromtheScientificAmericanarticleanddiscussingthepre-labquestionseitherasaclassorinsmallgroups.StudentswillthencarryouttheDNAisolationexperiment,andthenfinishtheperiodwithafinaldebrieftodiscusstheresults,thinkofwaystovarytheexperiment,andmakeconnectionstocurrentresearchintheglycomicsfield.
Activities/ProcedureforPart2:Pre-teaching,doneinadvanceofthislesson:
• Partsofacell,componentsandstructureofDNAGrade11&12Biology:DNAExtractionInquiry(fullperiod)Pre-labquestions:
1. Whatisthepurposeofaddingthedetergenttothemashedupfruit?ThepurposeofthedetergentistobreakdownthecellwallandnuclearmembranetoreleasetheDNA.Inregulareverydaylife,weusedetergenttobreakupthefatswhenwashingdishes.Itdoesthesamethinghere,breakingupthephospholipidbilayerofthecellularmembrane.
2. Whatisthepurposeofaddingthesalttothemashedupfruit?Itneutralizesthechargeofthephosphate,makingitmorehydrophobicinwaterandhelpsittoseparatebetter.
LastUpdate:April,2020
3. Whatisthepurposeofaddingcoldalcohol?DNAprecipitatesatthefruit/alcoholinterface.
4. NowthattheDNAisextracted,whatfurthertestingcanbedonewithit?OnceDNAisreleasedfromthenucleus,onecancutuptheDNAwithenzymesand(theoretically)dogelelectrophoresistoseparatethepiecesofDNA.
Directionstotheteacher:ReadoverthefollowingarticletogetanideaofoneprocedurefortheDNAextractioninquiry.https://www.scientificamerican.com/article/find-the-dna-in-a-banana-bring-science-home/
1. Showthestudentshowtodothegeneralsetup.ChallengethestudentstooptimizethisDNAextraction.YouareinanothercountryandyouhavetodoaDNAextractionaspartofastudyabroadprogram.SomematerialsthatyoufindeasilyinCanada(coffeefilters,strawberries)atthistimeofyeararenotsoeasytofind.Insteadoffeelingdefeated,youchallengeyourselftocompletethetaskwithothermaterialsthatmightbearoundthathavethesamefunction.Canyoustilldotheextraction?
2. Havestudentsbrainstormwhattheycanuseinsteadofcoffeefiltersandstrawberries.Encouragethemtothinkabouttheimportantaspects.Coffeefiltersstrainthingssothatsmallerparticlesstayinthefilterandtherestgoesthrough.Someanswersmayinclude:
Alternativestocoffeefilters
• Stockings• Meshcutfromalaundrybagusedfordelicates• Meshusedforcoveringsomefruit• Laboratoryfilters• Cheesecloth
Alternativestostrawberries(softfruit)
• Kiwis• Dragonfruit• Mango• Cannedfruit(pureedmango)• Peaches• Nectarines
LastUpdate:April,2020
Alternativestodishdetergent• Shampoo• 2in1shampoo• handsoap• canchangetheamountofdetergent• canchangetheamountofsalt
SummaryAsaclass,studentsview“TheGlycoNetStory”videotogetthecontextbehindcarbohydrateresearchandwhyitissoimportantforhumanhealth(therapeuticproteins,biofilms,etc.)andenergyneeds(biofuels).Individually,theylistentoandanswerquestionsaboutdifferentresearcherstalkaboutthebiochemicalanalysisthattheydo.Techniquessuchasgelelectrophoresis,TLC(ThinLayerChromatography)andFPLC(FastProteinLiquidChromatography)arediscussed.Afterthisintroduction,studentsreadaboutthecontextandimpactofusingDNAanalysisinthecourtroom.Thefollowingperiod,studentscontinuewiththisthemebydoingaDNAExtractionInquiry.
Assessment:Computerfile,“DNAExtractionInquiryBiology11&12SR(studentresource);”thenarrativereportoutlineandrubricfromtheinkchromatographylab(Grade9)canbeusedforthisactivityaswell.
Extensions:
1. DNADaywithCurioCity–DNAandFoodSecurity:http://curiocity.archive.letstalkscience.ca/Explore/ArticleId/5321/dna-day-2017.aspx
2. UsethesamepartA(videoclips)andPartB(literacypiece)butdoawaterpurificationinquiryandconnectto11UChemistry(SolutionsandSolubility)withconnectionstothemanyissuessurroundingwaterpurification,treatmentanddistributionaroundtheworld.
3. StudentscandouseGelElectrophoresis(Grade12Biology),usingavirtuallabfirst
andthenawetlab.
4. Whilestudyingphotosynthesis,insteadofdoingtheusualspinachchromatographylab,youcandotheseparationofthepigmentsinautumnleavesasavariation.HereisalabactivityintendedforhomeusefromScientificAmerican.https://www.scientificamerican.com/article/bring-science-home-leaf-colors/
LastUpdate:April,2020
5. Readaboutthe“BiochemistryofAutumnColours”onthefamousProfessor
Shakashiri’sScienceisFunwebpage:http://scifun.org/chemweek/PDF/Fall_Colors.pdforthehighlevelcontentfoundhere:https://staoblog.org/2014/10/13/world-science-festival-the-biochemistry-of-autumn-colors-world-science-festival/
References
• NakitaBeunbrazo,July2017.PresentationforTDSB(Powerpointpresentation).Wakarachuklaboratory,RyersonUniversity.
• Dr.WarrenWakarchuk,July2017.PresentationforTDSB(Powerpointpresentation).Wakarachuklaboratory,RyersonUniversity.
LastUpdate:April,2020
NarrativeLaboratoryReport1
Rationale:Theideabehindanarrativelaboratoryreportistomovestudentsbeyondaquestionandanswerstyleofanalysisorevenaformallaboratoryreport.Theyhavetoaskthemselvesthecorequestions:WhatdidIlookfor?HowdidIlookforit?WhatdidIfind?Whatdiditmean?
Itishopedthatbyaskingstudentstostepbackandtakeawiderviewoftheirwork,theywillmovetowardsopeninquirywheretheybegintowonderandgeneratetheirownquestionstoanswer(andbeingcomfortablewiththat).Aftertheinitialuncertaintyaboutmarksandsteppingoutoftheircomfortzone,studentsgenerallylikethefreedomtoworkinthisway,ifthetopiccaptivatesthem.Itistheresponsibilityoftheteachertoselectopportunitiesforthestudentsthathaveaninterestingcontextorsomeaspectthatisfascinating.
WhatwasIlookingfor?(Describeyourresearchquestionherealongwithyourhypothesis)
• Shouldbe1-2sentences,includesatestablequestion&hypothesisHowdidIlookforit?(Describeyourmethod)
• Shouldbereproducibleandstep-by-stepWhatdidIfind?(Showanyobservationsthatyouhad)
• Shouldincludetables(succinct&organizedwithheadings)andgraphs(labelaxes,includetitle,legendifnecessary)
Whatdoesthismean?(AnalysisandConclusion)• Shouldincludeanalysisofscientificobservationsanddata;shoulddiscussresults
andwhytheyoccurred• Writteninparagraphform
1ModifiedfromLenapeDistrictHighSchool’sNarrativeLabReport:https://www.lrhsd.org/cms/lib/NJ01000316/Centricity/Domain/26/templatepercent20narrativepercent20lab.pdf
LastUpdate:April,2020
RubricforNarrativeReport
Level1 Level2 Level3 Level4WhatwasIlookingfor?
Researchquestiondidnotincludeaproperlytestablequestion
Researchquestionwassomewhatfocusedwithatestablequestion
Researchquestionwaswellfocusedwithatestablequestion
Researchproblem/questionwashighlyfocusedwithatestablequestion
HowdidIlookforit?
Methodwasincomplete
Methodwasmostlythere
Methodwaswritteninagenerallyreproducibleway
Methodwaswritteninadetailedandhighlyreproducibleway
WhatdidIfind?
Observationswerepresentbutwerenotorganized/succinct;graphslackingaxes,labels&title
Observationsweresomewhatorganized&succinctwithtableheadings,goodgraphs
Observationswerewellorganized&succinctwithtableheadings,excellentgraphs
Observationswerehighlyorganized&succinctwithtableheadings,excellentgraphs
Whatdoesthismean?
Analysisdidnotfocusonthekeyquestionanddidnotaddressscientificerror
Analysissomewhatcapturedthekeyquestionsbutdidnotaddressthescientificerrorwell
Analysisgenerallycapturedthekeyquestionsandaddressedmostofthescientificerror
Analysiscapturedthekeyquestionsandworkedinscientificerrorinahighlevelway
LastUpdate:April,2020
AnswerKeyPartA:ListentotheScientistsTheGlycoNetStoryvideo:https://www.youtube.com/watch?v=CQGEGIoqTpQ
Letthevideoplay.Stopatthe1minutemark.1. Whatistheimportanceofunderstandingglycomics(thestudyofsugars)?
Communicationbycellsorinvasionintocellsisoftenmediatedbysugars.Ifweunderstandglycomics,wecaninterferewithinfectionprocesses.
2. WhatisGlycoNet?
GlycoNetisapan-Canadiannetworkofresearchersthatformedin2015todevelopsolutionstounmethealthneeds.
3. Whybringtheseresearcherstogether?Thispoolingofeffortsshouldresultinastreamlinedprocesstogetproductstomarketaswellascompetemoreeffectivelyontheinternationalstage.
Playvideofrom1min–2:474. WhatkindofgoodcanresultfromtheseGlycoNetpartnerships?
Anythingrangingfromnewvaccinestonewmedicinesfordiseasesthatarecurrentlyuntreatable.
5. Whatareashavetheymadeprogressin?Influenzaanddiabetes
MeettheGlycoNetScientistsfromRyersonUniversity,WakarchukLaboratory.Gotowww.glyconetchromatography.weebly.comVideoclip#1:Tasnim1–GraduateStudent
1. WhatisTasnim’sareaofresearch?Herareaofresearchisenzymaticsynthesis.Sheattachescertaincomponentsontoproteins,carbohydratesorotherbiomoleculesthatthelabneeds.
2. WhywouldTasnimfluorescentlylabel/tagmolecules?Researchthis.Itisnotmentionedinthevideobutwhenyouaretryingtopurifyamolecule,itisbeneficialtobeabletoseethedesiredproduct.Inthiscase,sheisvisualizingthemoleculeswithafluorescentdye.
LastUpdate:April,2020
Videoclip#2:Tasnim2–AtypicaldayforTasnimAbukar
1. TasnimmentionsthatsheusesTLC(ThinLayerChromatography)onatypicalday.WhatisTLC?Whatisitusedfor?TLCisusedtoseparatemixturesintoindividualcomponents.Withpaperchromatography,aninkcanbeseparatedintotheparticlesthatcomposeit.Forexample,ifpurpleinkweredottedontoapieceoffilterpaperanddippedintowateroranothersolventsuchasalcoholoracetone,thesolventwouldmoveupthepaperthroughcapillaryactionandcarrytheinkparticleswithit.Sincetheinkparticlesdifferinsomeway(size,charge,attractiontothepaper,etc.),theymovedifferentamountsandcanbeseenseparately.Formoreinformation,refertohttp://chemguide.co.uk/analysis/chromatography/thinlayer.html.
2. WhatdidTasnimsaywasthebasisoftheseparationintheTLC?
Shesaidthatthemoleculeswouldseparatebasedonpolarity.3. TasnimmentionedthatanothertechniquethatsheusesalotisFPLC(fastproteinliquid
chromatography).Whatisthis?Chromatographyinvolvestheseparationofmixturesintotheirindividualcomponentsbasedondifferentpropertiesofthecomponents.ItisusedalotinbiotechnologyandissimilartoHPLC(highperformanceliquidchromatography)butatahighpressure.Theseparationcouldbeduetosize,chargeorhydrophobicinteraction.Reference:http://fplc.weebly.com/background.html
4. Whatiselution?
Inproteincolumnchromatography,asolutioncontainingthedesiredprotein(plusimpurities)ispouredthroughthecolumn.Theproteinwillsticktothematerialinsideofthecolumnandtheimpuritieswillflowthrough.Later,anothersolutionwillbepouredthroughtoreleasethedesiredprotein,whichisthencollectedinpureformforfurtheruse.Thisprocessof“stripping”thecolumniscalledelutionandiscommonlyusedinbiotechnologyandbiochemistry.Reference:https://en.wikipedia.org/wiki/Elution
Videoclip#3:RayMartinez-Rodriguez-Summerstudent(1:10minutes)
1. WhatdoestheenzymethatJoseworkswithdo?
Itmovessugarsfromadonormoleculetoanacceptormolecule.2. Whataretwoproblemswiththisprotein(enzyme)?
LastUpdate:April,2020
Oneproblemisthatitishardtopurifyandtheotheristhatitisnotveryactive.3. JosedescribeshowaHistagcanhelpfixoneofthetwoproblemsmentionedabove.
Whatdoesithelpwith?DoanInternetsearchforHis-tag(histidinetag)purificationandIMAC(immobilizedmetalaffinitycolumn).Hementionsusinganickelcolumn.DrawapictureofanIMACcolumnandexplain.Scientistsaddhistidinetoproteinstohelptotagthemforapurificationprocess.Whentryingtoscaleupasubstanceforlargescaleproduction,itisimportanttobeabletoidentifyandpurifythefinalproduct.Thishelpswiththepurificationprocess.Seequestion#4fortherestoftheanswer.
4. Immobilizedlactaseinacalciumalginategelandpouringmilkthroughittobreakdown
thelactaseisasimilarideatotheIMACmentionedabove.Howarethesetwoproceduressimilarandhowaretheydifferent?TherepeatingHis(histidine)taghasacertainaffinityformetalssuchasnickelwhichareembeddedinthecolumn.Asamplethatishistidine-taggedcanbepouredthroughtheNicolumn,stickingtothecolumnwhileimpuritiesareflushedout.Thehis-taggedsamplecanbelaterrecoveredbyelutingwithanothersubstancesuchasimidazole,whichcompeteswithhistidineforthenickel.Inthisway,arecombinantproteincanberecovered.Thisisdifferentfromusingimmobilizedlactasesincelactosepassesthroughandtheenzymebreaksitdown.Noelutingisnecessarysincethegoalistogetridoftheoffendinglactosebybreakingitdown.Thebreakdownproductsarestillpresentinthelactose-freemilk.Thetwoprocessesaredifferentbecausethegoalsaredifferent.IMACusesaffinitytoremoveacertainmoleculefromothermoleculesinthelysate.Immobilizedlactasecarriesoutahydrolysisreactionononecomponentofthemixtureandthenreleasestheproductsbackintosolution.Thetwoprocessesaresimilarbecauseinbothcases,amixtureofsubstancesispouredthroughthecolumn.WithIMAC,onecomponentisremoved.Withimmobilizedlactase,lactoseisremovedbybreakingitdownintoothermolecules.Reference:“HowdoesHis-tagpurificationwork?”http://www.bio-rad.com/featured/en/his-tag-purification.html
Videoclip#4:Dr.TingDu-PostDoctoralFellow(26seconds)
1. Dr.Dutalksaboutpurifyinghertargetprotein.Howdoesshedothat?ShedoesthiswithFPLC(fastproteinliquidchromatograph)orAKTA(aproteinpurificationsystem).
LastUpdate:April,2020
2. Shealsotalksaboutconstructinganoperon.Whatisanoperon?Anoperonisaclusterofgenesthatcodesforaparticularproteinthatisregulatedbyasinglepromoter.
3. Whatisthefunctionoftheproteinsintheoperonthatsheistalkingabout?
TheseenzymesputanO-glycan(chainofcarbs)ontohertargetprotein.References:1.http://fplc.weebly.com/uses.html.2.https://www.britannica.com/science/operon
Videoclip#5:LauraKell,ResearchTechnician(58s)AsLauratalksaboutherjob,shementionstwomainresponsibilities.Oneofthemistrainingandmentoringotherteammembersandassistingthemtopushtheprojectsalong.
1. Isitsurprisingtoyouthataresearchtechnicianhasthisresponsibilityaspartoftheirjob?Answerswillvary.
2. Whatistheotherpartofherjob?
HerresearchincludesusinglabtechniquessuchasproteinpurificationusingFPLC(aformofliquidchromatographytoanalyzeandpurifyproteins).Shealsousesenzymeassayswithfluorescentlylabeledsubstrate(amoleculethatisacteduponbyanenzymetoproduceaproduct)whicharereactedwithenzymeswhicharethendevelopedonaTLC(thinlayerchromatography)plate.
3. Lauradescribeshertypicaldayandsays,“Well,thereisnotypicalday.”Doesthisappeal
toyou?Wouldyoulikeajobwheretherearedifferentthingshappeningday-to-dayorsomethingmoreconsistent?Answerswillvary.
4. Whatisanenzymeandsubstrate?Researchthis.
Anenzymeisaproteinthathelpstobreakdownorbuildupothermolecules.Asubstrateisamoleculethatanenzymeactsupontocreateaproduct.Anexampleistheenzymelactase,whichhelpstobreakdownthedoublesugarlactose(asugarfoundinmilk)intosinglesugars.
Clickonthe“LabTechniques”Tabatthetopofthewebsitepage.YouwillseesomeoftheRyersonUniversityscientistsatwork,demonstratinglabtechniquessuchas:gelelectrophoresis,TLCandSPE(solidphaseextraction).
LastUpdate:April,2020
ViewtheTLCdemonstrationvideo.Summarizetheconceptin1-2sentences.Summary:TLCworksbyseparatingamixtureintoitsindividualcomponentsbytheirproperties.
PartB:LiteracyConnectionReadtheJune9,2017CNNarticle“TheShiftingScienceofDNAintheCourtroom”byChannonHodge:http://www.cnn.com/2017/06/09/health/dna-technology-forensic-evidence/index.htmlorConnecttoGrade11GeneticsandBiodiversitybyreadingtheAugust28,2014ScienceNewsforStudentsarticlebyAlisonPearceStevens,“SavingtheBanana.”https://www.sciencenewsforstudents.org/article/saving-bananaOntheLines:MainIdea,Who?Whathappened?Include2definitionsofkeywordsAnswerswillvary.BetweentheLines:Why?Benefits?Consequences?Answerswillvary.
BeyondtheLines:Doyouagree?Howdoesthisaffectyourcommunity/city/country?Answerswillvary.