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Thediauxicgrowthcurve–Homeworkassignment

NextsessionwewilltrytoreproducethefamousdiauxicgrowthcurveexperimentfirstperformedbyJacquesMonod inthe1940’sduringhisPhDwork.SubsequentworkbyMonodledhimtodecipherthebasicmechanismsofgeneregulationandproposethelacoperonmodelof geneexpression, forwhichhe received theNobelprize togetherwithJacobandLwoffin1965.Theideabehindthediauxicgrowthexperiment(literallymeaningdoublegrowth)isverysimple–weputE.coli inamediumwithtwotypesofsugars, one that it can directlymetabolize (for example glucose) and a second sugar(lactoseforexample)thatneedstobefurtherprocessedinordertobemetabolizedbythecell.WhatMonoddiscoveredwasthatthecellpreferstofirstdigestglucose,whichendowsthecellwithahighergrowthrate(makingitmorefit),andonlywhenglucoseisexhausted from the medium will it start to synthesize the machinery needed totransportanddigestandultimatelygrow,albeitmoreslowly,onthesecondsugar.Thusgrowthonamixtureofsugarsthereforeexhibitsthefollowingphases: (1) fastgrowthonglucose (2) lagperiod (3) slowergrowthon lactose (4) saturation.See forexampleFig.1dtakenfromMonod’sNobellecture.ThefollowingisMonod’sownaccountofhisfindings.

lag

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“IwasworkingthenattheoldSorbonne,inanancientlaboratorythatopenedonagalleryfullofstuffedmonkeys.DemobilizedinAugustintheFreeZoneafterthedisasterof1940,IhadsucceededinlocatingmyfamilylivingintheNorthernZoneandhadresumedmyworkwithdesperateeagerness.Iinterruptedworkfromtimetotimeonlytohelpcirculatethefirstclandestinetracts.Iwantedtocompleteasquicklyaspossiblemydoctoraldissertation,which,underthestronglybiometricinfluenceofGeorgesTeissier,Ihaddevotedtothestudyofthekineticsofbacterialgrowth.Havingdeterminedtheconstantsofgrowthinthepresenceofdifferentcarbohydrates,itoccurredtomethatitwouldbeinterestingtodeterminethesameconstantsinpairedmixturesofcarbohydrates.Fromthefirstexperimenton,Inoticedthat,whereasthegrowthwaskineticallynormalinthepresenceofcertainmixtures(thatis,itexhibitedasingleexponentialphase),twocompletegrowthcyclescouldbeobservedinothercarbohydratemixtures,thesecyclesconsistingoftwoexponentialphasesseparatedbya‐completecessationofgrowth(Fig.1).”–1965NobellecturebyMonod

The following is a simple exercise in gross estimation. Monod concluded that whenglucoseisexhaustedbythecellthediauxicshiftoccurs.Canyoupredictbasedonsimpleargumentswhenthiswilloccur?Hereistheexperimentalsetup.At t=0 you inoculate a 1L growthmedium containing 0.1g/L of glucosewith 10mL ofsaturatedE.coliculture(@1.5∙108cells/mL)andallowthecellstogrowaerobicallyat37degC.Assumethatthecellsaregrowingexponentiallywithadoublingtimeof20min,andthatatthisgrowthrateanaveragecellhasabout6∙106proteinspercell(onaverage300aainlengtheach).Youmayalsoassumethatthemediumhasbeensupplementedwithaminoacids so that the cell doesn’tneed to synthesize thesebuildingblocks foritself.Undertheseconditionsitisknownthatmostoftheenergyconsumedbythecellisrequiredforaminoacidpolymerization(i.e.makingproteins).Finallyyouwillneedtoknowthatunderaerobicgrowthconditionsthecellcangenerate30ATPmoleculesfromeachglucosemolecule, and that4of theseATPmoleculesare required toelongateasinglepeptidebond.Hint:estimatethetotalnumberofglucosemoleculesinthemediumand compare that with the number of glucose molecules being consumed by theexponentiallygrowingpopulationofcells.Please also read up a little on the lac operon (for example in Alberts, Essential cellbiologypp.271‐275orPBoCpp.138‐140,section4.4.3)

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SomereferencesDiauxicgrowthhttp://nobelprize.org/nobel_prizes/medicine/laureates/1965/monod‐lecture.htmlhttp://en.wikipedia.org/wiki/Diauxiehttp://en.wikipedia.org/wiki/Lac_operonhttp://bioinfo.bact.wisc.edu/themicrobialworld/regulation.htmlBrock,Biologyofmicroorganisms,2006,pp.216‐218“TheGrowthofBacterialCultures”–Monod,AnnRevMicroBiol3:371,1949.“Thephenomenonofenzymaticadaptation”–Monod,GROWTH11(4):223‐289,1947GrowthphasesBrock,Biologyofmicroorganisms,2006,pp.142‐144Microbe,Schaechter,IngrahamandNeidhardt,2006,pp.55‐58,259‐262.Thelacoperonhttp://en.wikipedia.org/wiki/Lac_operonMicrobe,Schaechter,IngrahamandNeidhardt,2006,pp.229‐232.LittleAlberts,pp.271‐275PBoCpp.138‐140,section4.4.3Other:“BacterialGrowth:ConstantObessionwithdN/dt”–Neidhardt,JBact181:7405,1999.