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Proceedings of the National Academy of Sciences Building Biological Memory by Linking Positive Feedback Loops
Building Biological Memory by Linking Positive Feedback Loops
Dong-Eun Chang, Shelly Leung, Mariette R. Atkinson, Aaron Reifler, Daniel Forger, Alexander J. Ninfa and Clyde A. Hutchison IIIयह पुस्तक आपको कितनी अच्छी लगी?
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Proceedings of the National Academy of Sciences of the United States of America
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Building Biological Memory by Linking Positive Feedback Loops Author(s): Dong-Eun Chang, Shelly Leung, Mariette R. Atkinson, Aaron Reifler, Daniel Forger, Alexander J. Ninfa and Clyde A. Hutchison III Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 1 (Jan. 5, 2010), pp. 175-180 Published by: National Academy of Sciences Stable URL: http://www.jstor.org/stable/40536247 Accessed: 28-11-2015 12:19 UTC REFERENCES Linked references are available on JSTOR for this article: http://www.jstor.org/stable/40536247?seq=1&cid=pdf-reference#references_tab_contents You may need to log in to JSTOR to access the linked references. Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. National Academy of Sciences is collaborating with JSTOR to digitize, preserve and extend access to Proceedings of the National Academy of Sciences of the United States of America. http://www.jstor.org This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions positive Building biologicalmemory bylinking feedbackloops Dong-Eun Chang8, Shelly Leung3, Mariette R. Atkinson3,Aaron Reifler3,Daniel Forgerb,and Alexander J. Ninfa3'1 aDepartmentof Biological Chemistry,Universityof Michigan Medical School; and bDepartmentof Mathematics and Center for Computational Medicine and Biology,Universityof Michigan,Ann Arbor, Ml 48109 Edited by Clyde A. HutchisonIII,The J.Craig Venter Institute,San Diego, CA, and approved November 20, 2009 (received for review July23, 2009) ofactivator totheconc; entration protein promoter transcriptional thanone(8, 10a kinetic orderorsensitivity mustdisplay greater are met,bistability requirements maybe 12). If theseminimal conditions. to occurundersomeenvironmental anticipated of ofa bistable Animportant consisting genetic system example a singlepositivefeedbackloop was providedby theworkof efwhostudiedtheso-called"preinduction NovickandWeiner, coli(13). Thelac opfect"ofthelacZYAoperoninEscherichia whentheoperonis induced is observed eronpreinduction effect inas gratuitous known xenobiotic inducers, bynonphysiological but bind to the the cell can not metabolized that are ducers, by Lad repressor proteinand causeit to releasethelac operator thata effect refersto theobservation DNA. The preinduction wasrequired forthe inducer ofgratuitous concentration higher to maincellsthanwasrequired induction ofnaive(uninduced) inducedstate.At certainconcellsin thefully tainpreinduced as themaintenance known centrations ofinducer, concentrations, inducedcellsreandpreviously naivecellsremained uninduced, mainedintheinduced state,indefinitely (13).Evenwhencultures thelacZYAoperon levelof induction, showedan intermediate cells(13,14).Aloffintheindividual waseitherfully on orfully bistability| genetic network | syntheticbiology | ultrasensitive | hysteresis effect lacZYAoperonpreinduction experithoughtheoriginal and with TMG as the a chemostat mentswereperformed using ofexpression inducer, can thattheeffect information weshowinthesupporting displaya discontinuity geneticsystems Bistable stablesteadystatesare obtained be demonstrated cultures and with states,wheretwodistinct flask-grown usingstandard without thepresenceof stableintermediate hasconsteadystates.The IPTG as theinducer modeling (Fig.SI). Mathematical which stablesteady firmed determines of the in thelacZYAoperonand of bistability themechanism system history previous stateis occupied.One of the important andhighsensitivity feedback theroleofbothpositive problemsin systems confirmed and (14-17). Thissystem is established howgenetic istounderstand and remainsthemostwell-characterized bistability biology This is becausebistablegeneticswitchesplay an widely usedexampleofcellularbistability. regulated. suchas cellular The laçZYAoperonpreinduction ofcellularprocesses, roleina variety is due to thepositive effect important cellcycle,andthe feedback theeukaryotic on itsown oscillators, ofthelacYproduct, through progression permease, galactoside cellandtissuetypesin organisms expression ofdifferentiated inducer development (13,18).Thispermeaseallowsthegratuitous to thehuman(1-7). to enterintothecell.Whencellslackthegalactoside fromthetemperate bacteriophage ranging permease, a givencircuit as inthenaivestate,a highconcentration studieshavefocusedon whether is inducer ofgratuitous Manyprevious forsomerangeof required to display hasthecapacity oftheoperon, forinduction. bistability topology However, uponinduction thepossi- the cells come to acquiremanymoleculesof the galactoside conditions environmental (e.g.,reís.8-10).Although thatthe permeasethatcanbringaboutfurther isimportant, itisalsoimportant ofbistable behavior internalization ofthein- z bility at whichit occursbe large ducer(positivefeedback).The presenceof permeaseprotein conditions rangeof environmental control ofbiological toachievepractical Here, molecules a highintracellular cellstomaintain processes. allowstheinduced enough oftheparameters concentration andmanipulation wefocusuponidentification conevenwhentheextracellular of theinducer, atwhichbis- centration conditions therangeofenvironmental thatcontrol effectis is low.As expected,the lac preinduction tobe capableofbistability.eliminated known forsystems is obtained of thelacY gene(18). Furthermore, tablity uponmutation We use themethodsof synthetic wherethe underconditions is minimized biologyto createmodelex- thepreinduction effect ofmultiple to addressthefunctions of isdown-regulated positive function oftheLacY protein systems perimental (19).Inhibition feedback loopsinbistability. havearguedthattheminimal studies Theoretical requirements theremustbe sometype Author contributions:D.-E.C, D.F., and A.J.N.designed research; D.-E.C, S.L, A.R., and aretwofold. forgenetic First, bistability of positivefeedback Examplesof D.F. performed research; M.R.A. contributed new reagents/analytictools; D.-E.C, S.L, controlling geneexpression. itsown D.F., and A.J.N.analyzed data; and D.-E.C, D.F., and A.J.N.wrote the paper. drives an activator are when feedback protein positive of linked an even number orwhen negative regulatoryThe authors declare no conflictof interest. expression This article is a PNAS Direct Submission. the a blocks as when such are expression repressor steps present, orderor Freelyavailable online through the PNAS open access option. ofitsownexpression. ofa repressor Second,thekinetic mustbe 1To whom correspondence should be addressed. E-mail:aninfa@umich.edu. element feedback tothepositive ofthesystem sensitivity in thesimplecase wherea tran- This article contains supporting informationonline at www.pnas.org/cgi/content/full/ high(8, 10-12)..For example, theresponse ofthe 09083141 07/DCSupplemental. itsownexpression, activator drives scriptional A common topology found in many bistable genetic systemsis two interactingpositive feedback loops. Here we explore how this relativelysimple topology can allow testabilityover a large range of cellular conditions. On the basis of theoretical arguments, we predictthat nonlinear interactionsbetween two positive feedback loops can produce an ultrasensitive response that increases the range of cellular conditions at which bistabilityis observed. This predictionwas experimentallytested by constructinga synthetic genetic circuitin Escherichiacoli containingtwo well-characterized positivefeedback loops, linkedin a coherentfashion.The concerted action of both positivefeedback loops resulted in bistable behavior over a broad range of inducer concentrations;when either of the feedback loops was removed, the range of inducerconcentrations at whichthe systemexhibited bistabilitywas decreased by an order of magnitude. Furthermore,bistabilityof the system could be tuned by alteringgrowth conditionsthat regulate the contribution of one of the feedback loops. Our theoretical and experimental work shows how linked positive feedback loops may produce the robust bistable responses required in cellular networks that regulate development,the cell cycle,and manyother cellularresponses. UJ www.pnas.org/cgi/doi/10. 1073/pnas.09083 141 07 PNAS | January 5, 2010 This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions | vol. 107 | no. 1 | 175-180 occurswhenthePTS componentand Results theLacY permeaseactivity protein EIIAglc is present in its un- A GraphicAnalysisof the Problem.To understandthe factors signal-transduction stateand bindsto LacY (20). This occurswhen controlling of theactivatormoduleof the therangeofbistability phosphorylated the cell is grownin the presenceof PTS sugars,such as glucose, Atkinsonet al. oscillator,we use rate-balanceplotsfollowing the and, to lesserextents,whenthe cell is grownin the presenceof workof Ferrelland Xiong (10, 11). In thismethod,the ratesof othersubstratesthatexertcataboliterepression(21). Thus, the activatorproductionand decay are plottedas a functionof accells tivatorconcentration. effectwas notdiscerniblein glucose-grown lac preinduction Because activatorhas no directeffecton cells, the lac preinduction its own decay,we expectactivatordecay to be a simplelinear (Fig. SI). Even in succinate-grown in an experiment effectwas a fairlyweak bistability; usingIPTG functionof activatorconcentration(Fig. 1). Conversely,bioas the inducer,the range of inducerconcentrationsat which chemicalstudiesoftheactivationof transcription bytheNRI ~ P was observedwas narrow[about4-foldin flask-grown activatorindicateda bistability high kineticorder and an S-shaped reto cells (Fig. SI)]. Indeed, in flask-grown cells, it was difficult sponse (25) (Fig. 1). Steadystatesare possible onlywherethe of IPTG (Fig. SI). demonstrate a maintenanceconcentration curvesforproductionrateof activatorand decayrateofactivator In nature,simplebistablesystemswitha singlepositivefeed- intersect (Fig. 1). A keyfeatureof thegenetictoggleswitchused back loop are rarelyencountered;instead,naturalsystemsare in our studies is that activatorand repressorcompete for the complex and contain multiplefeedback loops that could be promoterthat drives transcription of the activatorstructural director indirectand usinga varietyofbiochemicalmechanisms, In this geneticsystem,activatorhas no direct gene (Fig. L4). mechanisms.Fourteen effecton the Lad in combination withadditionalregulatory repressor,which is presentat a low conexamplesof such systemshave been noted by Brandmanet al. stitutivelevel. The inducerIPTG servesto decrease the con(22). For example, in the genetic systemthat controlspro- centrationof functionalrepressor.Thus, changesin the IPTG gressionthroughthe cell cycle,the mitotictriggerproteinCdc2 concentration the curveforpromaybe thoughtof as shifting participatesin threepositivefeedbackloops (Cdc2 -» Cdc25 -» ductionof activatorto the the as or left, depictedin Fig. 1. right Cdc2; Cdc2-IWee-l-l Cdc2; Cdc2-IMytl-ICdc2) (22). Similarly, The at whichthe systemdisplays of IPTG concentrations range fortraversalof the startof the cell cyclein buddingyeast,the is thuslimitedto the extentto whichthe production Cdc28 protein participatesin two positive feedback loops bistability curve be shiftedto therightor theleftwhilestillmaintaining may (Cdc28-I Sicl-I Cdc28; Cdc28 -+ Cln -» Cdc28). Other systems at least two withthe decaycurve(Fig. 1). pointsof intersection include those responshowingmultiplepositivefeedbackloops In the systemconsideredabove,activatorproductionratedismammalian sibleforp53 regulation, Xenopusoocytematuration, a highsensitivity, whereasactivatordecayratewas linear, calciumsignaltransduction, eukaryoticchemotaxis,B cell fate played in accordance with the systemwe use. However,it experimental EGF receptorsignaling,blood clotting,and plaspecification, a noted that nonlinear should be degradationrate could having telet aggregation(noted in ref.22). The Bcl2 apoptoticswitch This result was also achieve bistability. presentedbyFerrell providesan additionalexample,wheretwoindependentpositive and help who considered a transduction system(26). signal Xiong, feedbackloops participatein producingbistability (23). Simpleinspectionof Fig. 1 revealstwo of the keyparameters Foundational work on the behavior of biological systems therangeofinducerconcentrations at whichthesystem affecting containingmultiplefeedbackloops was presentedby Thomas will The firstof these is the steepness(sensidisplaybistability. and D'Ari (24). Thisworkshowedhow thepresenceof multiple kinetic order) of the activatorproductioncurve; the feedbackloops could lead to an unexpectedly large numberof tivity, thisresponsecurveis,thegreateritmaybe shiftedto the steeper states. steady twointersections withthe left or the rightwhilestillmaintaining Two additionalhypothesesforthe presenceof multipleposthe rangeof intimescalesof the loops decay curve.A second keyparameteraffecting itivefeedbackloops are that different at whichbistability is observedis the abducer concentrations (22) provideforresistanceto noise undercertaincircumstances solute magnitudeof the activator'seffecton itself.That is, the robustto certainparametervariations(23). or make bistability Ferrellhas noted thatcoherentlinkageof a positivefeedback absoluteheightof theS-shapedactivatorproductioncurvelimits thedistanceit maybe displacedto theleftor therightwhilestill loop and a double-negativefeedbackloop in a systemof opwiththe decay curve.In over a wide range of maintainingat least two intersections posingenzymescould resultin bistability conditions(10). In naturalsystemswithcomplexcircuitarchi- this work,we focus on controllingthe steepnessof the protectures,testingtherolesof themultiplepositivefeedbackloops ductioncurve.In supportof the simplegraphicmethodused in and muchof theworkin thisarea to date has been Fig. 1, a more formalanalysisof our specificsystemleadingto is nontrivial the same conclusionsis presentedin the SI Text. theoretical. purely We developedan experimental multiloopsystembycombining the "activatormodule" of the syntheticgenetic oscillatorof HowCanthe Steepness oftheActivatorProductionCurveBe Increased? of a promoterto its activatoris dependentupon Atkinsonet al. (12Jwiththegalactosidepermeasefeedbackloop The sensitivity ofthelacZYA operon.The activatormoduleoftheAtkinsonet al. numerousfactors,such as the oligomericstate of the activator oscillator,when placed into cells that expressLad repressor protein,the numberof molecules that are requiredfor tranformsa genetictoggleswitchin whichan activator scriptionalactivation,and the details of the interactionsof constitutively, the formof theglnG product(NRI or activatorand polymerasewitheach otherand withothermacprotein, phosphorylated oftheglnGstructural gene,and romoleculesthatinteractwiththemand thereforecompetefor NtrC),activatesthetranscription to adjustin a Lad repressestranscription of theglnG gene. Because activator them.In mostcases, theseparametersare difficult and repressorcompeteforcontrolof transcription of theglnG systematic way and thus a general solutionto the problemof withthe level of IPTG re- increasingthe apparentkineticorderof an activator'seffecton gene, the systemdisplayshysteresis, of induction itselfmustemploya different approach.It has longbeen known quiredforinductiondependenton thepriorhistory of thissystemwas moreprominent thanthat thatultrasensitive responsesto a stimulusmay be obtainedin (12). The hysteresis systemsconsistingof linkedcyclesof reverdisplayedby the galactosidepermeasesystem,in thatbistability signaltransduction whenthestimulusregulatesmultiple was observedover an «10-fold range of IPTG concentrations siblecovalentmodification, (12). We showthatthemultiloopsystemobtainedbylinkingthe distinctactivitiesin the signalingsystem(27) (reviewedin ref. is referredto as "multistepultraactivatormodule and galactosidepermeasefeedbackloops dis- 28). Such ultrasensitivity could be sensitivity" played extensivebistability, indicatingthat bistability (27). Recently,Rossi and colleagues used a model builtup bylinkageof distinctfeedbackloops. systemto showthattheapparentkineticorderof a experimental 176 | www.pnas.org/cgi/doi/IO.IOyB/pnas.OSOSBMIO? This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions Chang et al. Fig. 1. Design and functionof the genetic toggle switch. (A) Basic circuitdesign for the genetic toggle switch.The Lacl repressorwas produced fromthe naturalwild-typelad gene. The activatormodule containingthe glnG structuralgene was located in the rbs region of the E. coli chromosome. The activator and repressorcompeted forcontrolof the expressionof the activatormodule promoter;when neitheractivator nor repressorproteinswere present,a weak promoter(not depicted) allowed for transcriptionof the activator gene (12). The reporterconsisted of a fusion of the activator-dependentglnK promoter withthe lacZ gene, located in the trp region of the chromosome. Forthe systemas shown with a single positivefeedback loop, the lacY gene was nextto lad, but contained a null mutation. (B) Graphicrepresentationof the factorsaffectingthe range of IPTGconcentrationsat which bistabilityis obtained. All of the plots depict rate of activatorproductionor destructionvs. the concentrationof activator.The destructionof activatoris not regulated by activatorand thus is likelyto have a slope of 1, as depicted. Conversely,the production of activator is known to display high kineticorder and is depicted as the S-shaped curve. The role of IPTG is to shiftthis S-shaped curve to the rightor the left,as depicted. Steady states occur when the two curves intersect,as indicated by small circles.The dotted circlein the Center plot depicts an unstable steady state. Factorscontrollingthe shape of the S-shaped activator productioncurve,such as the steepness of this curve or its absolute height/controlthe range of bistabilityof the system. responseto a stimuluswas increasedwhen the transcriptional stimulusaffectedboth the activationand the repressionof a promoter,relativeto the situationswherea singlefunctionwas regulated(29). We regardthisresultas a special class of multiand reasonedthatwe couldsimilarly increase stepultrasensitivity the apparentkineticorder of activator'sresponse to itselfby havingactivatorinfluencenotonlytheactivationof itsstructural ungene,but also the repressionof its own gene. Furthermore, like the systemof Rossi et al. (29), whichrequiresspecifically engineeredproteins,we soughtto have activatorfunctionto This was accomdecrease repressionof the systemindirectly. plishedby havingactivatordrivethe expressionof the galactoside permease that brings about the internalizationof the therepressor.In thebriefformalanalysis inducerthatinactivates of our specificsystempresentedin SI Text,we observedthatby of repression,the of activatorto inhibition theactivity extending alreadyhighkineticorderof the responseto activatorcould be increased. significantly FeedbackLoopsto Createa Composite Well-Characterized Combining SystemwithCoherentLoops.The experimental systemwithtwo each witha single feedback and two control systems loops, positive positivefeedbackloop,is depictedin Fig. 2. We used a previously describedchromosomally integratedfusionof the E. coli glnK genes of thelacZYA operon(30). Expromoterto thestructural pressionfromtheglnKpromoteris dependentupon theactivator NRI ~ P. The glnKpromoter-lacZYA ofthegenetictoggleswitch, suchthatthenoveljointcorrespondedto fusionwas constructed startcodon of the lacZ gene. Thus, in the rethe translational combinant operon,themajorlac operonoperatorelement(lacOl) was notpresentand neitherwas the minorlacO3 operator.The minorlacO2 operatorwas present,as thiselementis foundwithin intheabsenceofthemajor thelacZ structural gene.Nevertheless, lac operator(lacOl), itis anticipatedthatLacl repressorwillhave Changet al. littledirecteffect on transcription ofthelacZYA structural genesin the recombinantcontext,whereas expressionshould be tightly controlledbyactivator. We chosetheglnKpromoterand thisspecificoperon fusionforour experiments because priorworkhas basal expression inthe shownthatthisoperonfusionhas negligible absenceoftheactivator protein(30). Aversionoi'hzglnKp-lacZYA an internaldeletionwithinthelacY gene operonfusioncontaining was generatedbyrecombineering (31). To providethemaximum for thegenetictoggleswitch our stability synthetic geneticsystem, (activatormoduleof theAtkinsonet al. clock) was incorporated intotheE. coli chromosomein therbsregion,as describedpreviously(12). The activatorofthissystemrequiresphosphorylation foractivity, and we providedthisfunction byincludingwithinthe cell an alteredkinase proteinthatbringsabout the phosphoru ylationof activatorregardlessof nitrogenstatus.To provideacontrolsystemlackingthegalactosidepermeasepositivefeedback loop, we simplyused theversionof the systemwiththelacY null mutation.To providea controlsystemlackingthe positivefeedback loop of the activatormodule,but containingthe positive feedbackloop based upon galactosidepermease,additionalgewererequired(Fig. 2B). For thispurpose,we neticmanipulations createda systemin whichthelacZYA promoterregion(fromthe initiationcodon of upstreamlacO3 sitethroughthetranslational thelacZ gene) was fusedto theactivatorstructural gene.Thiswas placed intotherbsregionof the chromosome,analogousto the and combinedincellswith ofthegenetictoggleswitch, positioning theglnKp-lacZYAfusion,creatinga systemwhererepressorconwithactitrolof thelac promoterregulatedactivatorexpression, vatorthendrivingexpressionoflacZYA. i Synergyof PositiveFeedbackLoops. To measure the range of was obtained,cultures at whichbistability inducerconcentrations were incubatedfor12-14 h in the absence of induceron in the presence of saturatinginducer. The cells were then washed PNAS | January 5,2010 | vol.107 | no. 1 | 177 This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions Fig. 2. Strong bistabilitywas obtained by linkingdistinctpositive feedback loops. (A) Genetic toggle switch with a single positive feedback loop. (Left) Schematic depiction of the genetic systemin which the genetic toggle switchdrivesthe expression of the lacZYA operon, but the lacY gene contains a null mutation. (Right) Resultof bistabilityexperiment,showing an ~1 2-fold range of IPTGconcentrationsat which the systemdisplayed bistability.Symbols:0, naive (uninduced) culture; Q, preinduced culture. (B) Genetic systemwith a single positive feedback loop based on galactoside permease. (Left) Schematic of lacZYA. The lacY product,galactoside permease, provides positivefeedback byfacilitatingthe uptake of IPTG,which inactivatesrepressor.(Right)Resultof bistabilityexperiment,showing an ~4-fold range of inducerconcentrationsat which the systemdisplayed bistability.Symbolsare as in A. (C) Genetic system with two positive feedback loops. (Left) Schematic depiction of the genetic systemin which the genetic toggle switch drivesthe expression of the lacZYA operon, as in A except with a wild-typelacY gene. (Right) Result of bistabilityexperimentshowing that bistabilitywas obtained over an ~480-fold range of / IPTG. For A-C, cells were grown in minimalmedium with succinate as the carbon source and glutamine as the nitrogensource. and diluted 1 millionfoldinto freshmedium conthoroughly of inducer.These cultureswere tainingvariousconcentrations thengrownto midlogphase and the level of the lacZ product, ß-galactosidasewas measured(32). The greatestcontribution of the positivefeedbackloops is expectedin our systemwhenthe cells are grownon mediumin whichcataboliterepressionis minimizedand thusthegalactoside For thispurpose, permeaseloop is operatingwithoutinhibition. we used succinate-basedminimalmedium.Under these conditions,the systemwitha singlepositivefeedbackloop in which activatordrivesits own transcription over a displayedbistability 12-foldrangeofinducerconcentrations with (Fig. 24), consistent earlierobservations(12). The controlsystemlackingthepositive feedbackoftheactivatoron itsowntranscription, butcontaining a singlepositivefeedbackloop formedby galactosidepermease, over an ~4 -foldrange of inducerconcendisplayedbistability trations(Fig. 2B), similarto thebistability observedforthenative lacZYA system(Fig. SI). Remarkably,under these same conditions,the systemwithtwo functionalpositivefeedbackloops switch," (strainDE1010), whichwe referto as the"double-toggle over an ~480-foldrangeof inducerconcendisplayedbistability trations(Fig. 2C). Thus,thetwopositivefeedbackloops displayed powerfulsynergyin increasingthe range of inducer concentrationsoverwhichbistability was observed. The bistability of the double-toggle switchcould be controlled byincreasingcataboliterepression.For a verymodestdegreeof in the succataboliterepression, we includedcasein hydrolysate cinate growthmedium.Under these conditions,the range of IPTG concentrations at whichthesystemwas bistablewas ~ 100fold (Fig. 3/4).Strongercataboliterepressionwas obtainedby usingmediumcontainingboth glucose and casein hydrolysate; undertheseconditionstherangeofIPTG concentrations atwhich the systemwas bistablewas only~25 -fold(Fig. 3B). Apparently, cataboliterepressioncould be used to controlthecontribution of the galactosidepermease feedbackloop. Even thoughthe galactoside permease feedbackloop, when actingalone, did not in thepresenceof glucose(Fig. SI), bistability displaysignificant in thecontextofthedouble-toggle switchthisloop stillincreased therangeofbistability about ~2-foldin mediumcontainingboth This can be discernedby comglucose and casein hydrolysate. parisonwiththecontrolstrainwitha nullmutationinlacY grown underthe same conditions(Fig. 3C). Discussion We observed that distinctpositivefeedbackloops employing different biochemicalmechanismscould be linked to provide and thatthefunctions ofsuchsystems powerfulgeneticbistability were scalable byfactorsthataffectedone of thefeedbackloops. 178 | www.pnas.org/cgi/doi/10.1073/pnas.09083 14107 This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions Chang et al. Fig. 3. instabilityof the double-toggle switchwas tunable by growth substrates causing catabolite repression. (A and B) The double-toggle switchstrain depicted in Fig.2Cwas examined in medium containingsuccinate and casein hydrolysate(>A)and in medium containingglucose and casein hydrolysate(B). (C) The experimentis as in B, but the straincontained a null mutation in lacY and thus had only a single functioningpositive feedback loop. could be built up piecewise,by the coherent Thus, testability linkage of biochemicallydistinctfeedback mechanisms.Furbecause the different feedbackloops interactedwith thermore, each otherin a nonlinearway(in ourcase,bycausingan increase in the sensitivity of the responseto activator),fairlyweak feedback loops acted synergistically to produceextensivebistability. These observationprovidea generallyapplicablefoundationfor The therationalengineering ofsynthetic geneticbistablesystems. withnumerousinteracting prevalenceofcomplexnaturalsystems feedbackloops, whichregulatecriticalresponsessuch as progressionthroughthecell cycle,circadianclocks,or development, by may reflectthis capacityto build stronggeneticbistability collaborationof multipleweak elements.The sharpstate transitionsinvolvedin clocks,cell cycles,and irreversible morphogenic pathwaysare likelyto requiregeneticbistabilityover a broadrangeof conditions(e.g., refs.7 and 22), whichcould not evolvein a singlestepand had to be builtup piecewise. the pathTo developnovelsynthetic geneticbistablesystems, wayfollowedbynaturecouldbe mimickedbydevelopingcircuitry wheredifferent feedbackloops act coherently. Our workshows thatthereis no necessityforusinghighlyengineeredproteins; affecttranscriptional activationor represfactorsthatindirectly sion are just as good as factorsthat directlyinteractwiththe Thus,forexample,in ourcase DNA, as longas theyare effective. we wouldexpectthata circuitwhereactivatorbroughtabout the repressionof repressorsynthesis (alongwiththe activationof its own synthesis) would also bringabout verydramaticbistability. Such a systemwouldbe closerto thatused byRossi (29) thanis of using indirect the systemused in this paper. The flexibility the methods,suchas regulationofthepermeasethatinternalizes the inducer,followsthe pathwayused bybacteriaand simplifies of systems. engineering 1. Pomerening JR, Sontag ED, Ferrell JE, Jr (2003) Building a cell cycle oscillator: Hysteresisand bistabilityin the activation of Cdc2. Nat Cell Biol.5:346-351. 2. Sha W, et al. (2003) Hysteresisdrives cell-cycletransitions in Xenopus laevis egg extracts.Proc Nati Acad Sci USA 100:975-980. 3. Wang X, Hao N, Dohlman HG, Eiston TC (2006) Bistability,stochasticity,and oscillationsin the mitoqen-activatedprotein kinase cascade. BiophysJ 90:1961-1978. 4. FerrellJE,Jr(2002) Self-perpetuatingstates in signal transduction:Positivefeedback, double-negative feedback, and bistability.CurrOpin Cell Biol 14:140-148. 5. Bagowski CP, Ferrell JE, Jr (2001) Bistabilityin the JNK cascade. Curr Biol 11: 1176-1182. 6. Dubnau D, LosickR (2006) Bistabilityin bacteria. Mol Microbiol 61:564-572. 7. TysonJJ,Chen KC, Novak B (2003) Sniffers,buzzers, toggles and blinkers:Dynamicsof regulatoryand signaling pathways in the cell. CurrOpin Cell Biol 15:221-231. 8. Angeli D, FerrellJE,Jr,Sontag ED (2004) Detection of multistability, bifurcations,and hysteresisin a large class of biological positive-feedbacksystems.Proc Nati Acad Sci USA 101:1822-1827. 9. CraciunG, Tang Y, Feinberg M (2006) Understandingbistabilityin complex enzymedrivenreaction networks.Proc Nati Acad Sci USA 103:8697-8702. Chang et al. We arguethatall ofthecases ofbistablesystems withmultiple interacting geneticfeedbackloops noted above maybe demonthatwas stratinga formof the same multistepultrasensitivity studiedin signaltransduction systems25 years ago (27, 28). A and well-understood limitation in genetic to bistability significant forthe critical systemsis obtaininga sufficiently highsensitivity regulatorystep, such as the activator'seffectupon its own expressionin our system(8, 10-12). We expectthatthistypeof geneticmultistepultrasensitivity mayplay an importantrole in geneticregulationrequiringa highkineticorderin those cases where multiplefeedbackloops are focused on controllingthe expressionof a gene. Methods Genetic Elements.The activator module of the NC12 syntheticgenetic clock and the fusion of the glnK promoter to the lacZYA structuralgenes were described previously(12, 30). The fusion of the lacZ promoter to the glnG structuralgene was constructedin several steps, as described in SI Text,and the primersused for constructionof the lacY null mutation by recombineering are listed in SI Text. All molecular cloning, PCR, P1virtransduction, and plasmid transformationused standard techniques (33, 34). The bacterial strainsused and their relevant genotypes are listed in Table S1. PhysiologyExperiments.Growthmedium for bistabilityexperimentsused Wsalts (12), with added vitaminB1 (0.004% wt/vol),tryptophan(0.04% wt/vol), and glutamine (0.2% wt/vol),and contained succinate at 0.4% wt/vol,casein hydrolysateat 0.5% wt/vol,and glucose at 0.4% wt/vol,as indicated. IPTG was used at 0.4 mM for overnightinductionof the cultures.ß-Galactosidase activitywas measured using the method of Miller (32). ACKNOWLEDGMENTS.We thank PatrickO'Brien for reviewingan earlyversion of this manuscript.This work was supported by GrantGM63642 (to AJ. N.) from the National Institutes of Health-National Institute of General Medical Sciences. 10. FerrellJE,Jr(2008) Feedback regulation of opposing enzymes generates robust,allor-none bistable responses. CurrBiol 18:R244-R245. 11. Xiong W, FerrellJE,Jr(2003) A positive-feedback-basedbistable 'memory module' that governs a cell fate decision. Nature 426:460-465. 12. Atkinson MR, Savageau MA, Myers JT, Ninfa AJ (2003) Development of genetic circuitryexhibitingtoggle switch or oscillatorybehavior in Escherichiacoli. Cell 113: 597-607. 13. Novick A, Weiner M (1957) Enzyme induction as an all-or-none phenomenon. Proc Nati Acad Sci USA 43:553-566. 14. Ozbudak EM, Thattai M, Lim HN, Shraiman Bl, Van Oudenaarden A (2004) Multistabilityin the lactose utilization network of Escherichia coli. Nature 427: 737-740. 1.5. Santillán M, Mackey MC (2004) Influence of catabolite repression and inducer exclusion on the bistable behavior of the lac operon. BiophysJ 86:1282-1292. 16. Wong P, Gladney S, Keasling JD (1997) Mathematical model of the lac operon: Inducer exclusion, catabolite repression,and diauxic growth on glucose and lactose. Biotechnol Prog 13:132-143. PNAS | January 5, 2010 This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions | vol. 107 | no. 1 | 179 8 17. Yildirim N, Mackey MC (2003) Feedback regulation in the lactose operon: A mathematical modeling studyand comparison with experimental data. BiophysJ8A: 2841-2851. 18. Herzenberg LA (1959) Studies on the induction of beta-galactosidase in a cryptic strainof Escherichiacoli. Biochim BiophysActa 31:525-538. 19. Cohn M, Horibata K (1959) Inhibitionby glucose of the induced synthesisof thebetagalactoside-enzyme systemof Escherichiacoli. Analysisof maintenance. J Bacteriol 78:601-612. 20. Inada T, Kimata K,Aiba H (1996) Mechanism responsibleforglucose-lactose diauxie in Escherichiacoli: Challenge to the cAMP model. Genes Cells 1:293-301. 21. Hogema BM, et al. (1998) Inducerexclusion in Escherichiacoli by non-PTSsubstrates: The role of the PEP to pyruvate ratio in determiningthe phosphorylationstate of enzyme IIAGIc.Mol Microbio! 30:487-498. 22. Brandman O, FerrellJE,Jr,Li R, Meyer T (2005) Interlinkedfast and slow positive feedback loops drive reliable cell decisions. Science 310:496-498. 23. Cui J,Chen C, Lu H, Sun T, Shen P (2008) Two independent positive feedbacks and bistabilityin the Bcl-2apoptotic switch. PLoS ONE 3:e1469. 24. Thomas R, D'Ari R (1989) Biological Feedback (CRC, Boca Raton, FL). 25. Feng J,et al. (1992) Role of phosphorylatedmetabolic intermediatesin the regulation of glutamine synthetasesynthesisin Escherichiacoli. J Bacteriol 174:6061-6070. 180 26. FerrellJE,Jr,Xiong W (2001) Bistabilityin cell signaling: How to make continuous processes discontinuous,and reversibleprocesses irreversible.Chaos 11:227-236. 27. Goldbeter A, Koshland DE, Jr(1984) Ultrasensitivityin biochemical systemscontrolled by covalent modification.Interplaybetween zero-order and multistepeffects.J Biol Chem 259:14441-14447. 28. Koshland DE, Jr, Goldbeter A, Stock JB (1982) Amplificationand adaptation in regulatoryand sensorysystems.Science 217:220-225. 29. Rossi FM, KringsteinAM, Spicher A, GuicheritOM, Blau HM (2000) Transcriptional control: Rheostat converted to on/offswitch.Mol Cell 6:723-728. 30. AtkinsonMR, Blauwkamp TA, Bondarenko V, StuditskyV, NinfaAJ(2002) Activation of the ginA, glnK, and nac promoters as Escherichiacoli undergoes the transition from nitrogen excess growth to nitrogenstarvation.J Bacteriol 184:5358-5363. 31. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products. Proc Nati Acad Sci USA 97:6640-6645. 32. Miller JH (1972) Experimentsin Molecular Genetics (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY). 33. Sambrook J, FritschEF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Lab Press,Cold Spring Harbor, NY), 2nd Ed. 34. SilhavyTJ,Berman ML, EnquistLW (1 984) ExperimentswithGene Fusions(Cold Spring Harbor Lab Press,Cold Spring Harbor, NY). | www.pnas.org/cgi/doi/10.1073/pnas.0908314107 This content downloaded from 139.80.123.51 on Sat, 28 Nov 2015 12:19:27 UTC All use subject to JSTOR Terms and Conditions Chang et al.