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ThediauxicgrowthcurveHomeworkassignment

Nextsessionwewilltrytoreproducethefamousdiauxicgrowthcurveexperimentfirst performedbyJacquesMonodinthe1940sduringhisPhDwork.Subsequentworkby Monodledhimtodecipherthebasicmechanismsofgeneregulationandproposethe lac operon model of gene expression, for which he received the Nobel prize together withJacobandLwoffin1965.Theideabehindthediauxicgrowthexperiment(literally meaningdoublegrowth)isverysimpleweputE.coliinamediumwithtwotypesof sugars, one that it can directly metabolize (for example glucose) and a second sugar (lactoseforexample)thatneedstobefurtherprocessedinordertobemetabolizedby thecell.WhatMonoddiscoveredwasthatthecellpreferstofirstdigestglucose,which endowsthecellwithahighergrowthrate(makingitmorefit),andonlywhenglucoseis exhausted from the medium will it start to synthesize the machinery needed to transportanddigestandultimatelygrow,albeitmoreslowly,onthesecondsugar.Thus growthonamixtureofsugarsthereforeexhibitsthefollowingphases:(1)fastgrowth on glucose (2) lag period (3) slower growth on lactose (4) saturation. See for example Fig.1dtakenfromMonodsNobellecture.ThefollowingisMonodsownaccountofhis findings.

lag

IwasworkingthenattheoldSorbonne,inanancientlaboratorythatopened onagalleryfullofstuffedmonkeys.DemobilizedinAugustintheFree Zoneafterthedisasterof1940,Ihadsucceededinlocatingmyfamilyliving intheNorthernZoneandhadresumedmyworkwithdesperateeagerness.I interruptedworkfromtimetotimeonlytohelpcirculatethefirstclandestine tracts.Iwantedtocompleteasquicklyaspossiblemydoctoraldissertation, which,underthestronglybiometricinfluenceofGeorgesTeissier,Ihaddevoted tothestudyofthekineticsofbacterialgrowth.Havingdeterminedthe constantsofgrowthinthepresenceofdifferentcarbohydrates,itoccurredto methatitwouldbeinterestingtodeterminethesameconstantsinpaired mixturesofcarbohydrates.Fromthefirstexperimenton,Inoticedthat, whereasthegrowthwaskineticallynormalinthepresenceofcertainmixtures (thatis,itexhibitedasingleexponentialphase),twocompletegrowthcycles couldbeobservedinothercarbohydratemixtures,thesecyclesconsisting oftwoexponentialphasesseparatedbyacompletecessationofgrowth (Fig.1).1965NobellecturebyMonod

The following is a simple exercise in gross estimation. Monod concluded that when glucoseisexhaustedbythecellthediauxicshiftoccurs.Canyoupredictbasedonsimple argumentswhenthiswilloccur? Hereistheexperimentalsetup. At t=0 you inoculate a 1L growth medium containing 0.1g/L of glucose with 10mL of saturatedE.coliculture(@1.5108cells/mL)andallowthecellstogrowaerobicallyat 37degC.Assumethatthecellsaregrowingexponentiallywithadoublingtimeof20min, andthatatthisgrowthrateanaveragecellhasabout6106proteinspercell(onaverage 300aainlengtheach).Youmayalsoassumethatthemediumhasbeensupplemented with amino acids so that the cell doesnt need to synthesize these building blocks for itself.Undertheseconditionsitisknownthatmostoftheenergyconsumedbythecell isrequiredforaminoacidpolymerization(i.e.makingproteins).Finallyyouwillneedto knowthatunderaerobicgrowthconditionsthecellcangenerate30ATPmoleculesfrom each glucose molecule, and that 4 of these ATP molecules are required to elongate a singlepeptidebond.Hint:estimatethetotalnumberofglucosemoleculesinthemedium and compare that with the number of glucose molecules being consumed by the exponentiallygrowingpopulationofcells. Please also read up a little on the lac operon (for example in Alberts, Essential cell biologypp.271275orPBoCpp.138140,section4.4.3)

Somereferences
Diauxicgrowth http://nobelprize.org/nobel_prizes/medicine/laureates/1965/monodlecture.html http://en.wikipedia.org/wiki/Diauxie http://en.wikipedia.org/wiki/Lac_operon http://bioinfo.bact.wisc.edu/themicrobialworld/regulation.html Brock,Biologyofmicroorganisms,2006,pp.216218
TheGrowthofBacterialCulturesMonod,AnnRevMicroBiol3:371,1949.

ThephenomenonofenzymaticadaptationMonod,GROWTH11(4):223289,1947 Growthphases Brock,Biologyofmicroorganisms,2006,pp.142144 Microbe,Schaechter,IngrahamandNeidhardt,2006,pp.5558,259262. Thelacoperon http://en.wikipedia.org/wiki/Lac_operon Microbe,Schaechter,IngrahamandNeidhardt,2006,pp.229232. LittleAlberts,pp.271275 PBoCpp.138140,section4.4.3 Other:
BacterialGrowth:ConstantObessionwithdN/dtNeidhardt,JBact181:7405,1999.

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