Grade: Grade11/12 Subject: Unit: Title: Separation

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/


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)


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


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.


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


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/


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.


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


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


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.


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)?


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).


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).


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.

Documents

Grade: Grade11/12 Subject: Unit: Title: Separation