Engineered Biofilms for Materials Production and Patterning

Engineered Biofilms for MaterialsProduction and PatterningThe Harvard community has made this

article openly available. Please share howthis access benefits you. Your story matters

Citation Chen, Yuyin. 2016. Engineered Biofilms for Materials Productionand Patterning. Doctoral dissertation, Harvard University, GraduateSchool of Arts & Sciences.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493429

Terms of Use This article was downloaded from Harvard University’s DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA

http://osc.hul.harvard.edu/dash/open-access-feedback?handle=&title=Engineered%20Biofilms%20for%20Materials%20Production%20and%20Patterning&community=1/1&collection=1/4927603&owningCollection1/4927603&harvardAuthors=9cf7df5b37b974b9c58530c011530963&departmentBiophysics

http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493429

http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA



Engineeredbiofilmsformaterialsproductionandpatterning

Adissertationpresented

by

YuyinChento

TheCommitteeonHigherDegreesinBiophysics

inpartialfulfillmentoftherequirementsforthedegreeof

DoctorofPhilosophyinthesubjectof

Biophysics

HarvardUniversity

Cambridge,Massachusetts

Feburary10,2016

Copyright©2016YuyinChen

Allrightsreserved.

iii

Prof.TimothyK.Lu YuyinChen

Engineeredbiofilmsformaterialsproductionandpatterning

Abstract

Naturalmaterials, such as bone, integrate living cells composed of organic

molecules together with inorganic components. This enables combinations of

functionalities, such as mechanical strength and the ability to regenerate and

remodel,which arenotpresent in existing syntheticmaterials. Taking a cue from

nature,weproposethatengineered‘livingfunctionalmaterials’and‘livingmaterials

synthesisplatforms’thatincorporatebothlivingsystemsandinorganiccomponents

couldtransformtheperformanceandthemanufacturingofmaterials1.Asaproof‐

of‐concept,werecentlydemonstratedthatsyntheticgenecircuitsinEscherichiacoli

enabledbiofilms tobebotha functionalmaterial in itsownrightandamaterials‐

synthesisplatform.Todemonstratetheformer,weengineeredE.colibiofilmsintoa

chemical‐inducer‐responsive electrical switch. To demonstrate the latter, we

engineeredE.coli biofilms to dynamically organize biotic‐abioticmaterials across

multiple length scales, template gold nanorods, gold nanowires, and

metal/semiconductor heterostructures, and synthesize semiconductor

nanoparticles1.Thus, tools fromsyntheticbiology, suchas those forartificialgene

regulation, can be used to engineer the spatiotemporal characteristics of living

systemsandtointerfacelivingsystemswithinorganicmaterials.Suchhybridscan

possess novel properties enabled by living cells while retaining desirable

iv

functionalities of inorganic systems. These systems, as living functionalmaterials

and as livingmaterials foundries,wouldprovide a radically different paradigmof

materials performance and synthesis –materials possessingmultifunctional, self‐

healing, adaptable, and evolvable properties that are created and organized in a

distributed,bottom‐up,autonomouslyassembled,andenvironmentallysustainable

manner.

v

Tableofcontents

Title i

Copyright ii

Abstract iii‐iv

Tableofcontents v

Acknowledgements vi‐vii

Chapter1–Introduction:EngineeringLivingFunctionalMaterials 1‐13

Chapter2–Synthesisandpatterningoftunablemultiscalematerialswith

engineeredcells 14‐44

Appendix 45‐145

vi

Acknowledgements

Theprocessofputtingtogetherthisthesisprovidedanopportunitytoreflect

ontheperiodofintellectualandpersonalgrowththatIwasluckyenoughto

experienceoverthepastfewyears.

ItwasmytremendouslygoodfortunetohavejoinedProf.TimLu’slabjustas

itwasgettingofftheground,andtohaveaPIwhofacilitatedmygrowthboth

character‐wiseandintellectually.WorkingwithTiminculcatedinmegreater

decisivenessandbetterabilitytoprioritize,whichwillservemewellinallaspectsof

lifegoingforward.WorkinginTim’slabhasalsoshapedthewayIthinkabout

biologicalquestions;learningtheengineer’sapproachofmeasurement+

quantitativemodelingasawaytounderstandhasgivenmeapowerfulsetoftools

withwhichtodissectbiologicalsystemsthatIstudyinthefuture.

Iamverygratefultomycollaborators,labmates,andclassmatesfortheir

camaraderie:NicoleBillings,ZhengtaoDeng,EleonoreTham,MichelleLu,Rob

Citorik,SamPerli,AllenCheng,NateRoquet,ChaoZhong,UrartuSeker,andothers.

MicheleJakoulovandJimHoglegotogreatlengthstomaketheBiophysicsprogram

awarm,connectedplacedespiteusstudentsbeingspreadoutoverthreecampuses.

Ialsowishtothankfacultywhogenerouslytooktimefromtheirschedulesto

serveonmyDACandThesiscommittees:Profs.JimHogle,MarkusBuehler,George

Whitesides,DavidClarke,ShmuelRubinstein,andJenniferLewis.

TheHertzFoundation,OfficeofNavalResearch,andNIHsupportedme

financiallyduringgraduatetraining.IamespeciallygratefultotheHertzFoundation

vii

forcreatingacommunityoflike‐mindedindividualswho,byexampleoftheirblue‐

skiespursuits,providedreassurancethatitisOKtotakeintellectualrisks.

Myparents,HuiHuiandJingke,mysisterAnna,andmychildhoodfriendAlex

Flishavebeenabedrockofsupportthroughoutmyentirelife.Thegreatestblessing

ofallduringmygraduateschoolyearshasbeenmeetingmywife,Siting.Shehas

mademeawarmer,wiser,andlivelierperson,andIamoverjoyedthatweare

embarkingonalifetogether.

1

Chapter1Publication:thischapterisadaptedfrom

ChenAY,ZhongC,LuTK.Engineeringlivingfunctionalmaterials.ACSSynthBiol.

2015Jan16;4(1):8‐11.

Contributions:AYCandTKLwrotemaintext,CZprovidedinput,AYCmadefigures.

2

Anoverarchinggoal of syntheticbiology is todesignbiological systems foruseful

applications.Inexploringthespaceofpossibilities,onelogicalapproachistoconsiderthe

characteristicpropertiesoflivingsystems,howtheymaybeuseful,andhowsyntheticbiol‐

ogytoolscanengineerthem.Threesuchpropertiesareevolvability,self‐organization,and

responsiveness to environment. We propose that: 1) these properties of living systems

havebeenproductivelyapplied insyntheticbiology,2) thesecharacteristicsareusefulto

materials science by enabling new functionalities and synthesis capabilities, and 3) syn‐

theticbiologyiswell‐positionedtoharnessthesefeaturestocreatenovellivingfunctional

materialsandmaterialsfoundries(Figure1).

Proteinengineeringandheterologousexpressionhaveallowedproteindomainsto

beborrowedfromnatureandmodifiedorfusedinvariouscombinationstogenerateuseful

products,suchassoftmaterials.Forexample,pHandtemperature‐sensitiveleucinezipper

domainsfusedtohydrophilicdomainshaveledtopolymersthatreversiblyformahydro‐

gelinresponsetotheirenvironment1.Spidersilkproteinhasalsobeenrecombinantlyex‐

pressedandspunintofibers,withprocessingofthisaggregation‐proneproteinfacilitated

byextracellularsecretion2.

Traditionally,whenengineeredproteinsareusedasbuildingblocks formaterials,

only the first step in creating thematerials (i.e., heterologous expression of subunits) is

performedbycells.Assemblyofsubunitsintohigher‐orderstructuresisperformedinvitro

aftersubunitpurification.However,livingsystemsarecapableofmuchmorethanheterol‐

ogousproteinexpression.Asaresultofadvancesinsyntheticbiology,thereistheoppor‐

tunitytoevolveordesignmaterialssubunitsandtoengineercellstoassemblesubunitsin‐

3

tohigher‐ordermaterials,ratherthandependsolelyoninvitroassembly3.Havingcellsper‐

formassembly,orevenbeincorporatedaspartofthefinalmaterial,wouldopenupmany

newcapabilitiesfornovelmaterials,suchasself‐healing,remodeling,hierarchicalorgani‐

zation,andotherpropertiescharacteristicof livingsystems.Furthermore,suchmaterials

couldbegrowninbottom‐up,energyefficientprocesses,thusenablingdistributedmanu‐

facturing.Weenvisionthattheselivingfunctionalmaterialscouldrevolutionizethefunda‐

mentalwaysinwhichourworldmakesandusesmaterials.

Figure1‐1|Foundationalpropertiesoflivingsystemsgiverisetousefulpropertiesforma‐

terials functionandsynthesis. Integratingnon‐livingand livingsystemsenablesthecrea‐

tionof‘livingfunctionalmaterials’.

Evolvability

4

Livingsystemsareabletogenerateheritablediversityintheformofgeneticvaria‐

tionthatmanifestsasphenotypesthatcanbeacteduponbyselectivepressures.Thisprop‐

ertyhasbeenappliedinsyntheticbiologytocreatedirectedevolutionplatforms4thathave

beenusedtooptimizemetabolicpathways,createnovelenzymes,andevolveproteindo‐

mainswithusefulbindingproperties.

Anearlydirectedevolutionplatform,phagedisplay,hasprovenusefultomaterials

scienceasawaytoselectforpeptidesthatbindandnucleateinorganicmaterials.Thisap‐

proacheventuallyallowedfabricationofgeneticallyengineeredbatteries5andotherdevic‐

es.Newdirectedevolutionplatforms leveragingcells couldbeadapted tooptimizepath‐

waysforassemblingmaterials.Suchaplatformcouldallowtuningofnotonlythepeptide

sequenceandsubunitcompositionofmaterialssuchascollagen,polyesters,andpolysac‐

charides,butalsohowthesubunitsareassembled–animportantdeterminantofmaterial

properties.

Self‐organization

Living systems can perform impressive feats of self‐organization that are hierar‐

chical and coordinated in space and time, a prime example being embryogenesis. This

propertyhasbeenstudiedusingsynthetic‐biologyapproaches,aswellasappliedbroadly

forpatterningbasedonintercellularcommunicationandmotility6,7,forsynchronizationof

cellpopulations,andforintracellularorganizationtoachievecellpolarization.

Nature’s solutions for creating hierarchal self‐organized structures8 (biomole‐

culesbiomolecularassembliesorganellescellstissuesorgansorganisms)

5

maybemappedontotheproblemsthatmaterialsscientistsfaceinfabricatingmulti‐scale

patternedmaterials. Self‐organization in biology is carried out by genetic programs and

aided by physical forces; one‐dimensional strings of letters in DNA, encoding genes and

regulatory elements, can somehow direct the fabrication of complex three‐dimensional

structuresincellsandorganisms.

Understandingandmodifyingtheseprogramsthroughsyntheticbiologymayallow

us to harness them formakingmaterials that assemble autonomously. Early steps have

beentakeninthisdirectionatvariousscales.Workinbiomolecularself‐assemblyusesthe

physicalcomponentsthatstoreandcarryoutgeneticprogramstocreatenanoscalemateri‐

als.IntricatestaticanddynamicstructuresmaybepreciselyassembledbyusingDNAasa

structuralmaterialaswellasthecarrierofinstructionsforitsownassembly9;similarfeats

maybeachievedwithdesignerRNAandproteins.Self‐organizationatlargerscaleshasalso

been harnessed to create materials, some examples being: active self‐assembled matter

based on microtubules and kinesin10, self‐organized tissues11 and organoids12, and self‐

organizedrobotswarms13(inspiredbysocialinsects)thatcreateuser‐definedstructures.

Responsivenesstoenvironment

Living systems sense inputs from the environment, integrate them, and respond

with appropriate outputs. Environmental responsiveness is a property that is one of the

building blocks of evolvability and self‐organization, and is useful in its own right. The

sense‐and‐respondpropertyoflivingsystemshasbeenappliedbroadlyinsyntheticbiolo‐

gy14,15,someexamplesbeingoptogeneticcontrolofcellbehavior,light‐darkboundaryde‐

6

tection, sensing‐and‐destructionofpathogenicmicrobesviadetectionofquorumsensing

molecules, sensing‐and‐destruction of cancer cells via detection ofmicroRNA signatures,

sensing‐and‐ameliorationofhighuricacidlevels,andmulti‐cellularcomputationinwhich

cellsimplementingsimplelogicgatesarewiredtogetherbychemicalsignaling.Cellshave

also been incorporated into materials as sense‐and‐respond modules that, for example,

produceurateoxidaseinresponsetouricacidorproduceinsulinuponactivationbylight.

Living sense‐and‐respond systems can be useful as “smart” environmentally re‐

sponsive factories formaterialsor “smart” environmentally‐responsivematerials (if cells

themselvesareincorporatedaspartofthematerialandendowitwithfunction).Aspecific

exampleofaresponsivematerialwouldbeonethatsensesdamageandautonomouslyself‐

heals.Suchasystemwouldbecapableofsensingandrepairingmorediversetypesofdam‐

age compared to current non‐living self‐healingmaterials,which largely respond tome‐

chanicalandthermaldamagewithrelativelysimplefixes16.

Bridgingthefunctiongapwithinorganicmaterials

While livingmattermadeof organicmolecules offers exciting prospects fornovel

functionalmaterials, it canbedeficient incertainusefulproperties, suchas theability to

conductelectronsandtheabilitytointeractwithmagneticfields.Thisgapcanbebridged

by incorporating inorganic components that possess such properties, creating multi‐

functionalmaterialsthatcombinethecapabilitiesoflivingsystemswiththoseofinorganic

materials. An example fromnature ismagnetotactic bacteria,which are able to navigate

magnetic field linesbasedontheirabilitytobiomineralizeferromagneticcrystalsandor‐

7

ganizethemintochains(magnetosomechains).Inasyntheticcontext, interfacingbiology

with inorganicmaterials canbe accomplished via biomineralization,whichmaybe engi‐

neeredwithsyntheticbiology,orviabioconjugatechemistry.Asanexampleoftheformer,

gene clusters encoding the biosynthetic pathway for magnetosome chains in M.

gryphiswaldensehavebeentransferredtoaforeignmicrobialhosttoformfunctional,het‐

erologousmagnetosomes.Asanexampleofthelatter,semiconductordeviceswereincor‐

poratedintotheextracellularmatrixofengineeredtissuestogivehybrid,or“cyborg”tis‐

sues17.

Aplatformforengineeringlivingfunctionalmaterials

Asdescribedabove,alargebodyofliteraturehasdevelopedaroundengineeringliv‐

ingsystemsthatevolve,self‐organize,andcompute.Acomplementaryliteraturehasdevel‐

opedaroundengineeringinorganicnanomaterialswithdiverse,tunableproperties.Lever‐

agingthesecapabilities,werecentlypresentedanovelplatformforintegratinglivingcells

withinorganiccomponentsintolivingfunctionalmaterials18(Figure2).WeengineeredE.

colibiofilmswithartificialgenecircuitstocontrolthesynthesisofextracellularcurliamy‐

loid nanofibers. These nanofiberswere functionalizedwith peptide domains to interface

themwith inorganicmaterials.Thisplatformenablesmultiplenovelmaterialsproperties

derivedfromthecombinationofsyntheticbiologyandmaterialsscience.Specifically,these

engineeredE.colibiofoundriescanbeusedtomanufacturematerialswithcontrollablena‐

noscale‐to‐microscalestructureandcompositionbycontrollingeitherthetimingormagni‐

tude(e.g.,frequencymodulationoramplitudemodulation,respectively)ofexternalinduc‐

8

er inputs. Furthermore, by using cell‐cell communication circuits, the synthetic biofilms

generatedmaterialswhosenanoscale‐to‐microscale compositionandstructurewerevar‐

iedinadynamicandautonomousfashion,withnorequisiteuserinput.

Moreover,by incorporatingsyntheticgenecircuitswithother formsofpatterning,

such as spatial inducer gradients and protein engineering,wewere able to achieve self‐

organizationofmaterialsacrossmultiplelengthscaleswithbiofilms.Theabilitytohierar‐

chicallyorganizematerialsacrossmultiplelengthscalesisamajorchallengeformaterials

science, but is one that canbe readily addressedwithbiological systems. Specifically, by

combiningsyntheticgenecircuitswithspatialinducergradientstocontrolcurlinanofiber

production,engineeredbiofilmswereabletoimplementnanoscale‐to‐millimeter‐scalepat‐

terning.Byintegratingsyntheticgenecircuitswithproteinengineeringofthecurliamyloid

subunitsthemselves,theartificialbiofilmswereabletomorefinelycontrolthenanoscale‐

to‐microscalepatterningofthecurlinanofibers.Withfurtherintegrationoftop‐downpat‐

terningstrategies(suchasinputsofpatternedlightor3Dprinting)withbottom‐uporgani‐

zation(suchasrationaldesignofproteinself‐assemblyordirectedevolutiontoachievede‐

sired morphologies) and synthetic gene circuits, the precise, dynamic, and autonomous

controlofmulti‐scalestructureshouldbeachievablebylivingfunctionalmaterials.

Wealsodemonstratedthatlivingfunctionalmaterialsandlivingmaterials‐synthesis

platformsbased onbiofilms can organize and synthesize biotic‐abiotic hybrid structures

withnovel functions.Forexample,goldnanorods,nanowires,andchainsweretemplated

on biofilm‐synthesized curli nanofibers. This allowed the implementation of electrically

conductivebiofilmswitcheswhose conductivity couldbe toggledonwith theadditionof

9

external chemical signals. In addition, semiconductor quantumdotswere synthesized or

organizedonbiofilm‐fabricatedcurlinanofibers,andco‐assembledwithmetalnanoparti‐

cles. The resulting metal/semiconductor heterostructures exhibited modulated fluores‐

cenceproperties.Thus,wehaveshownthatbiofilm‐based living functionalmaterialscan

beusedtosynthesizeandorganizebiotic‐abioticsystems,aswellasmodulatetheirproper‐

ties.Sincebiofilmsarerobustandabundantlivingsystemsthatareeasytogrowandscale

up, they are promising substrates for the distributed, environmentally‐friendlymanufac‐

turingofmaterialsanddevices.Weenvisionthatrelatedplatformscouldenablethecrea‐

tion of cell‐synthesized batteries and solar cells, self‐healing living‐cell glues, self‐

regenerating catalytic membranes and filters, enzymatic scaffolds for tunable reaction

rates,anddynamicextracellularmatricesfortissueengineering.

10

Figure1‐2 |Alivingmaterials‐synthesisplatformandlivingfunctionalmaterialbasedon

E.colibiofilms(FigureadaptedfromChenetal.18).Thisstrategyisabletoa)patternmate‐

rials in response to external inputs, b) organize materials autonomously via cell‐to‐cell

communication,c)patternmaterialsacrossmultiple lengthscales,d) interfacewith inor‐

ganiccomponentstoenableelectricalconductivityandmodulatedfluorescence,ande)im‐

plementachemically‐responsiveelectricalswitchbasedonbiofilms.

11

Outlook

Living system properties of evolvability, self‐organization, and responsiveness to

the environment, in conjunctionwith tools to interface cellswith inorganic components,

constitute a foundation for synthesizingmaterials with novel functionalities. Our recent

workwithengineeredbiofilms18establishesaproof‐of‐conceptthatlivingcellularsystems

canactnotonlyas foundries fornewmaterials,butcanalsobe incorporatedintohybrid

materialssystems.Man‐madematerialsareoftenmadebycentralized,top‐down,energy‐

intensiveprocesses,andarelimitedintheirabilitytorespondandadapt.Theintegrationof

engineeredlivingsystemswithinorganiccomponentstocreate‘livingmaterials‐synthesis

platforms’and‘livingfunctionalmaterials’hasthepotentialtodramaticallychangethefab‐

ricationanduseofmaterials.

12

References

1 Petka, W. A., Harden, J. L., McGrath, K. P., Wirtz, D. & Tirrell, D. A. Reversiblehydrogels from self‐assembling artificial proteins. Science 281, 389‐392, doi:DOI10.1126/science.281.5375.389(1998).

2 Widmaier,D.M.etal.EngineeringtheSalmonellatypeIIIsecretionsystemtoexport

spider silkmonomers.Molecularsystemsbiology5, 309, doi:10.1038/msb.2009.62(2009).

3 Rice,M.K.&Ruder,W.C.Creatingbiologicalnanomaterialsusingsyntheticbiology.

ScienceandTechnologyofAdvancedMaterials15,014401(2014).4 Wang, H. H. et al. Programming cells by multiplex genome engineering and

acceleratedevolution.Nature460,894‐898,doi:10.1038/nature08187(2009).5 Lee, Y. J.etal. Fabricatinggenetically engineeredhigh‐power lithium‐ionbatteries

usingmultiplevirusgenes.Science324, 1051‐1055,doi:10.1126/science.1171541(2009).

6 Basu, S., Gerchman, Y., Collins, C. H., Arnold, F. H. & Weiss, R. A synthetic

multicellular system for programmed pattern formation.Nature434, 1130‐1134,doi:10.1038/nature03461(2005).

7 Payne, S. et al. Temporal control of self‐organized pattern formation without

morphogen gradients in bacteria. Molecular systems biology 9, 697,doi:10.1038/msb.2013.55(2013).

8 Weiner, S. & Wagner, H. D. The material bone: Structure mechanical function

relations. Annu Rev Mater Sci 28, 271‐298, doi:DOI10.1146/annurev.matsci.28.1.271(1998).

9 Seeman,N.C.NanomaterialsBasedonDNA.AnnuRevBiochem79, 65‐87,doi:DOI

10.1146/annurev‐biochem‐060308‐102244(2010).10 Sanchez,T.,Chen,D.T.,DeCamp,S.J.,Heymann,M.&Dogic,Z.Spontaneousmotion

in hierarchically assembled active matter. Nature 491, 431‐434,doi:10.1038/nature11591(2012).

11 Liu, J. S. & Gartner, Z. J. Directing the assembly of spatially organized

multicomponent tissues from the bottom up. Trends in cellbiology 22, 683‐691,doi:10.1016/j.tcb.2012.09.004(2012).

12 Lancaster, M. A. et al. Cerebral organoids model human brain development and

microcephaly.Nature501,373‐379,doi:10.1038/nature12517(2013).

13

13 Werfel, J., Petersen, K. & Nagpal, R. Designing Collective Behavior in a Termite‐

Inspired Robot Construction Team. Science 343, 754‐758,doi:10.1126/science.1245842(2014).

14 Benenson, Y. Biomolecular computing systems: principles, progress and potential.

Naturereviews.Genetics13,455‐468,doi:10.1038/nrg3197(2012).15 Brophy, J.A.&Voigt,C.A.Principlesof genetic circuitdesign.Naturemethods11,

508‐520,doi:10.1038/nmeth.2926(2014).16 Wool, R. P. Self‐healing materials: a review. Soft Matter 4, 400‐418,

doi:10.1039/b711716g(2008).17 Tian,B.etal.Macroporousnanowirenanoelectronicscaffoldsforsynthetictissues.

NatMater11,986‐994,doi:10.1038/nmat3404(2012).18 Chen, A. Y. et al. Synthesis and patterning of tunable multiscale materials with

engineeredcells.NatMater13,515‐523(2014).

14

Chapter2Publication:thischapterisadaptedfrom

ChenAY,DengZ,BillingsAN,SekerUO,LuMY,CitorikRJ,ZakeriB,LuTK.Synthesis

andpatterningoftunablemultiscalematerialswithengineeredcells.NatMater.

2014May;13(5):515‐23.

Contributions:TKLandAYCconceivedtheexperiments.AYCplannedall

experimentsandexecutedthemajorityoftheexperiments,ZDperformedquantum

dotsynthesisandaidedinHRTEMimagingforFig6,ANBperformedmicrofluidic

experimentsandfluorescencemicroscopyforFig1,MYLaidedincloning,andRJC

aidedinstrainconstruction,AYC,ZD,ANBandTKLanalyzedthedataanddiscussed

results.AYCwrotethemanuscriptandmadefigureswithinputfromTKL.

15

Naturalmulticellularassembliessuchasbiofilms,shells,andskeletaltissueshave

distinctivecharacteristicsthatwouldbeusefulformaterialsproductionandpatterning1‐9.

Theycandetectexternalsignalsandrespondviaremodelling,implementpatterningacross

differentlengthscales,andorganizeinorganiccompoundstocreateorganic‐inorganic

composites.Inthiswork,suchsystemsprovideinspirationforthedesignof

environmentallyresponsivesystemsthatcanintegratebioticandabioticmaterialsvia

hierarchicalself‐assembly.Toachievethesecapabilities,weengineeredartificialgene

circuitsandself‐assemblingamyloidfibrilstogetherwithsyntheticcellularconsortia10‐16

andabioticmaterials.

Ourmodelsystemiscurli,anextracellularamyloidmaterialproducedbyE.colithat

formsfibrilsbasedontheself‐assemblyofthesecretedmajorcurlisubunitCsgA17.

SecretedCsgAmonomersaretemplatedonCsgB,whichisanchoredtothecellsurface,to

formcurlifibrils;moreover,CsgAsecretedfromonecellcaninteractwithCsgBonother

cells17.Usingsyntheticriboregulators18,weimplementedinducibletranscriptionaland

translationalcontrolovertheexpressionofCsgAsubunitsengineeredtodisplayvarious

peptidetags,whichcaninterfacewithinorganicmaterials.Wetransformedoursynthetic

circuitsintoanE.coliMG1655PRO csgAompR234hoststrain(seeSupplementaryTable3

andSupplementaryFig.20),whichhastheendogenouscsgAgenedeleted.TheompR234

mutationenablescurliproductioninliquidmediaat30°Cbyenhancingtheexpressionof

genesfromthenativecurlioperon,includingcsgB19,20.Wefirstintroducedhistidine‐tagged

CsgA(CsgAHis)expressionundertightregulationbyananhydrotetracycline(aTc)inducer‐

responsiveriboregulator18(Fig.1a).CsgAHiscontainedtwohistidinetags,oneinserted

beforethefirstrepeatdomainandoneinsertedafterthelastrepeatdomaininCsgA

16

(SupplementaryTable1).TheresultingcellstrainwasdesignatedaTcReceiver/CsgAHis.

Immuno‐goldlabellingexperimentswithanti‐CsgAantibodies(M.Chapman,Universityof

Michigan21,22)showedthatcurlifibrilswereonlyproducedinthepresenceofaTc(Fig.1b

andSupplementaryFig.1).Usingconfocalmicroscopy,wecharacterizedbiofilmsformed

byaTcReceiver/CsgAHiscellsaugmentedwithanmCherry‐expressingplasmidforconvenient

visualization.ThisstrainformedbiofilmsonlywheninducedbyaTc,bothunderstatic

cultureconditions(Fig.1candSupplementaryFig.2a)andwhenculturedinmicrofluidic

flowcells(Fig.1dandSupplementaryFig.2b).Biofilmgrowthwasconfirmedwitha

standardcrystal‐violet(CV)assay(SupplementaryFig.3).Wealsoquantifiedcurli

productionwithdotblotsandfoundayieldof63±5.8(s.e.m.)mg/cm3ofbiofilmafter24h

(SupplementaryFig.22).

Tocreateengineeredcellularconsortiaformaterialspatterning,webuiltthree

additionalstrains:onewithCsgAunderregulationbyanacyl‐homoserinelactone(AHL)‐

inducibleriboregulator(AHLReceiver/CsgA),onewithCsgAunderregulationbyanaTc‐

inducibleriboregulator(aTcReceiver/CsgA),andonewithCsgAHisunderregulationbyan

AHL‐inducibleriboregulator(AHLReceiver/CsgAHis).Thesestrainsonlyproducedcurlifibrils

inthepresenceofthecognateinducer,demonstratingtightandorthogonalregulationof

csgAandcsgAHisexpression(SupplementaryFig.8).Moreover,insertionofheterologous

histidinetagsdidnotinterferewithcurlifibrilformationbasedonCongoRedassaysand

TEMimaging(SupplementaryFig.4and5).

17

Figure2‐1|Inducibleproductionofengineeredcurlifibrilsandbiofilms.a,

Riboregulatorcircuitstightlyregulateexpressionofcurlisubunits,suchasCsgAHis.

ProductionofCsgAHisrequirestheexpressionoftrans‐activatingRNA(taRNA).ThetaRNA

preventsthecis‐repressive(cr)sequencefromblockingtheribosome‐bindingsequence

(RBS)controllingtranslationofthemRNAtranscript.Intheabsenceofinducer,mRNAand

taRNAlevelsarelow,thusleadingtosignificantrepressionofgeneexpression.The

18

Figure2‐1(Continued)

additionofaTcinducestranscriptionofbothcsgAHismRNAandtaRNA,thusenabling

CsgAHisproduction.Tightregulationofcurliexpressionisusefulforcontrollingpatterning

(SupplementaryFig.19).b,Immuno‐labellingofcurlifibrilswithrabbitanti‐CsgA

antibodiesandgold‐conjugatedgoatanti‐rabbitantibodies.Positive‐control(“+ctrl”)

MG1655ompR234cells(“ompR234”,seeSupplementaryTable3),whichhaveanintact

endogenouscsgAgene,producecurlifibrilsthatwerelabelledbyanti‐CsgAantibodiesand

areattachedtocells.However,negative‐control(“−ctrl”)cellswiththecsgAgeneknocked

outandnocsgA‐expressingcircuits(“csgAompR234”,seeSupplementaryTable3),aswell

asaTcReceiver/CsgAHiscellsintheabsenceofaTc,didnotproducecurlifibrils.Inducing

aTcReceiver/CsgAHiscellswithaTcenabledthesynthesisofcurlifibrilsthatwerelabelledby

anti‐CsgAantibodiesandattachedtocells.Scalebarsare200nm.c,Confocalmicroscopy

andbiomassquantificationrevealedthatunderstaticcultureconditions,E.coliompR234

cellsformedthickadherentbiofilms.However,E.colicsgAompR234cells,aswellas

aTcReceiver/CsgAHiscellsintheabsenceofaTc,didnotformbiofilms.Inducing

aTcReceiver/CsgAHiscellswithaTcledtotheformationofthickadherentbiofilms.d,Confocal

microscopyandbiomassquantificationrevealedsimilarbiofilm‐formingcapabilitiesbyE.

coliompR234andinducedaTcReceiver/CsgAHiscellswhengrowninflowcells.Toenable

visualization,wetransformedaconstitutivemCherry‐expressingplasmidintoallstrains

(seeSupplementaryMethods).CellsweregrowninliquidM63mediawithglucose;the

correspondingexperimentsforothermediaconditionsareshowninSupplementaryFigure

1and2.Scalebarsinc)andd)are50µm,andorthogonalXZandYZviewsaremaximum‐

intensityprojections.

19

Externallycontrollablepatterning

WeengineeredconsortiacomposedofAHLReceiver/CsgAandaTcReceiver/CsgAHiscells

toproducetwo‐componentproteinfibrilscomposedofCsgAandCsgAHis(Fig.2).Bytuning

thepulselengthsandpulseamplitudesofAHLand/oraTc,fibrilswithdifferentstructures

andcompositionswereformed.Forexample,wemixedequalnumbersofAHLReceiver/CsgA

andaTcReceiver/CsgAHiscellstogetherandinducedthismixed‐cellpopulationfirstwithAHL,

followedbyaTc(Fig.2a).Inanalogytoblockco‐polymers,thisproducedblock“co‐fibrils”

consistingofblocksofCsgA(unlabelledfibrilsegments)andblocksofCsgAHis(fibril

segmentslabelledbynickelnitrilotriaceticacid‐conjugatedgoldparticles(NiNTA‐AuNPs).

NiNTA‐AuNPsspecificallylabelledCsgAHis‐basedcurlifibrilsbutnotCsgA‐basedcurli

fibrils(SupplementaryFig.9).

WetunedthelengthdistributionoftheCsgAandCsgAHisblocks,aswellasthe

relativeproportionsofCsgAandCsgAHis,bychangingtherelativelengthsofAHLpulses

versusaTcpulses.AsAHLinductiontimeincreased,non‐NiNTA‐AuNP‐labelledfibril

segmentsincreasedinlength,indicatinglongerCsgAblocks(Fig.2bandSupplementary

Fig.6a).Atthesametime,theproportionoffibrillengthlabelledwithNiNTA‐AuNP

decreased,indicatingahigherrelativeproportionofCsgAinthefibrils(Fig.2b).With

temporalseparationinexpression,thedistinctCsgAandCsgAHissegmentswithintheblock

co‐fibrilswerelongerthanthoseinco‐fibrilsassembledwhenCsgAandCsgAHiswere

secretedsimultaneouslywithnotemporalseparation,eventhoughtheoverallCsgAto

CsgAHisratiosweresimilar(SupplementaryFig.6a).Thus,engineeredcellscantranslate

20

thetemporalintervallengthofinputsignalsintodifferentnanoscalestructuresand

compositionsofmaterials.

Wealsotunedthelengthdistributionsofthetwotypesofblocks,aswellastheir

relativeproportions,byinducingsimultaneousexpressionoftheCsgAvariantswith

differentconcentrationsofAHLandaTc(Fig.2c).WithAHL‐onlyinduction,fibrilswere

almostuniformlyunlabelled;withincreasingaTcconcentration,thepopulationaswellas

lengthsofunlabelledfibrilsegmentsdecreasedwhilethoseoflabelledfibrilsegments

increased(Fig.2d,SupplementaryFig.6b).WithaTc‐onlyinduction,fibrilswerealmost

uniformlylabelledbyNiNTA‐AuNPs;withincreasingAHLconcentration,thepopulationas

wellaslengthsofunlabelledsegmentsincreased(SupplementaryFig.7).Thus,engineered

cellscantranslatetheamplitudesofinputsignals,suchasinducerconcentrations,into

differentnanoscalestructuresandcompositionsofmaterials.

21

Figure2‐2|Conversionoftimingandamplitudeofchemicalinducersignalsinto

materialstructureandcomposition.a,Induciblesyntheticgenecircuitscouplecurli

subunitsecretiontoexternalchemicalinducers.Engineeredcellscontainingthesecircuits

cantranslateinductionpulselengthintonanoscalestructureandcompositionofblockco‐

fibrils.b,WefirstusedAHLtoinducesecretionofCsgAfromAHLReceiver/CsgAandthen

usedaTctoinducesecretionofCsgAHisfromaTcReceiver/CsgAHis.Wetunedtherelativeblock

lengthsandproportionsofCsgAandCsgAHis(plotoftheproportionoffibrillengthlabelled

byNiNTA‐AuNP,solidgreyline)bychangingtherelativelengthsofAHLversusaTc

inductiontimes.Scalebarsare200nm.c,Syntheticgeneticregulatorycircuitsthatcouple

curlisubunitsecretiontoexternalinducersignalscantranslateinducerconcentrationinto

nanoscalestructureandcompositionofblockco‐fibrils.d,EngineeredcellsAHLinduced

secretionofCsgAfromAHLReceiver/CsgA,whileatthesametime,aTcinducedsecretionof

CsgAHisfromaTcReceiver/CsgAHis.Wetunedtherelativeblocklengthsandproportionsof

22


CsgAandCsgAHisbychangingtherelativeconcentrationsofAHLandaTcinducersapplied

simultaneously.Thesolidgreylineindicatestheproportionoffibrillengthlabelledby

NiNTA‐AuNPwithvaryingconcentrationsofaTcandconstant100nMAHL.Detailed

histogramscanbefoundinSupplementaryFigure6.Scalebarsare200nm.

Autonomouspatterning

Cellularcommunitiescontainingsyntheticcellularcommunicationcircuits23‐26can

autonomouslyproducedynamicmaterialswhosestructureandcompositionchangeswith

time(Fig.3).SinceE.colidoesnotnormallyproduceAHL,wefirstengineeredanE.coli

strainthatconstitutivelyproducesAHLandinduciblyproducesCsgAinthepresenceofaTc

(AHLSender+aTcReceiver/CsgA).ThisstraincommunicatedwithAHLReceiver/CsgAHiscellsviathe

diffusiblecellularcommunicationsignal,AHL.WethencombinedAHLSender+aTcReceiver/CsgA

andAHLReceiver/CsgAHiscellsinvaryingratios(Fig.3a).Inductionofthismixed‐cell

populationbyaTcresultedinCsgAsecretion.Overtime,AHLaccumulationledto

increasingsecretionofCsgAHis,thusgeneratinganincreasedpopulationandlengthsof

CsgAHisblocks,andahigherrelativeproportionofCsgAHisinmaterialcomposition(Fig.3b

andSupplementaryFig.10).Thetemporaldynamicsofchangesinmaterialcomposition

wastunablebytheinitialseedingratioofAHLSender+aTcReceiver/CsgAtoAHLReceiver/CsgAHis

cells(Fig.3b).WhenonlyAHLSender+aTcReceiver/CsgAcellswerepresent,fibrilswerealmost

23

uniformlyunlabelled;whenonlyAHLReceiver/CsgAHiscellswerepresent,nofibrilswere

formed(Fig.3b).

Figure2‐3|Syntheticcellularcommunicationfordynamic,autonomousmaterial

productionandpatterning.a,Syntheticgenecircuitsthatcouplecurlisubunitsecretion

toexternalinducersignals,whencombinedwithsyntheticcellularcommunicationcircuits,

allowfortheproductionofmaterialswhosestructureandcompositionchanges

autonomouslywithtime.AHLSender+aTcReceiver/CsgAsecretedbothCsgAandAHL.AsAHL

signalaccumulated,AHLReceiver/CsgAHissecretedincreasinglevelsofCsgAHis.b,Usingthe

24


autonomouscellularcommunicationsystem,thelengthofCsgAHisblocksandthe

proportionofCsgAHisincreasedwithtime(plotoftheproportionoffibrillengthlabelledby

NiNTA‐AuNP,greylines).Thisbehaviorcouldbetunedbytheratiooftheseedingdensity

ofAHLSender+aTcReceiver/CsgAcellstoAHLReceiver/CsgAHiscells.Whenonly

AHLSender+aTcReceiver/CsgAcellswerepresent,theresultingfibrilswerealmostuniformly

unlabelled;whenonlyAHLReceiver/CsgAHiscellswerepresent,nocurlifibrilswereformed

(Controls).DetailedhistogramscanbefoundinSupplementaryFig.10.Scalebarsare

200nm.

Multiscalepatterning

Inaddition,engineeredcellularconsortiacanachievespatialcontrolovermultiple

lengthscales.Geneticregulationofsubunitexpressionallowsfibrilpatterningfromtensof

nanometrestomicrometres,whilespatialcontrolatthemacroscalecanbeachievedvia

spatiallyvaryinginducerconcentrations.Thesetwomethodsofcontrolcanbecombinedto

creatematerialspatternedacrossmultiplelengthscales(Fig.4).Todemonstratethis,we

createdagarplateswithopposingconcentrationgradientsofAHLandaTcandoverlaid

bacterialpopulationsconsistingofequalnumbersoffourcellstrains:AHLReceiver/CsgA,

aTcReceiver/CsgAHis,AHLReceiver/GFP,andaTcReceiver/mCherry.TheAHLReceiver/GFPand

aTcReceiver/mCherrycellsenabledvisualizationofinducerconcentrationgradients(Fig.4b

andSupplementaryFig.12).AHLReceiver/CsgAandaTcReciever/CsgAHiscellssecreteddifferent

25

levelsofCsgAandCsgAHis,dependingontheirpositionsontheconcentrationgradient,to

generateaspatialgradientofchangingfibrilstructures(Fig.4a).Thismultiscalematerial

waspatternedatthenanoscaleasblockco‐fibrilsandatthemillimetrescalewithposition‐

dependentfibrilstructure(Fig.4bandSupplementaryFig.11a).Agarplateswithout

inducerconcentrationgradientsdidnotgeneratefibrilstructuresthatvariedalongthe

plate(SupplementaryFig.11a).

Proteinengineeringcanalsocontrolthestructureofcell‐producedbiomaterialsat

thenanoscale.WehypothesizedthatfusingtandemrepeatsofCsgAtogetherwould

increasethedistancebetweenequivalentpositionsonadjacentmonomerswhere

functionaldomainscanbedisplayed.ConcatenatingeighttandemrepeatsofCsgAand

addingahistidinetagtotheC‐terminus(8XCsgAHis)resultedinfibrilsthatwerelabelledby

asyncopatedpatternofNiNTA‐AuNP,withclustersofparticlesseparatedby33.3±27.1

(s.e.m.)nm(Fig.4candSupplementaryFig.11b).Usingthisfinding,wedemonstrateda

secondexampleofmultiscaleassembly.Specifically,wecombinedequalnumbersof

AHLReceiver/8XCsgAHisandaTcReceiver/CsgAHiscells.Weinducedthismixed‐cellpopulation

sequentiallywithAHLfollowedbyaTc(Fig.4d)togenerateblockco‐fibrilsconsistingof

8XCsgAHissegmentsandCsgAHissegmentspatternedacrossthenanometretomicrometre

scales(Fig.4dandSupplementaryFig.11c).

26

Figure2‐4|Multiscalepatterningwithcellularconsortiaviasyntheticgene

regulationcombinedwithinducergradientsandsubunitengineering.a,Synthetic

genecircuitsthatcouplecurlisubunitsecretiontoexternalinducersignals,when

combinedwithaspatialinducergradient,enablepatterningacrossmultiplelengthscales.

WeusedanagarplatewithopposingconcentrationgradientsofAHLandaTctoachieve

controlatthemacroscale(SupplementaryFig.12).Thiswascombinedwithregulationof

nanoscalepatterningtoachievemultiscalepatterning.Embeddedintopagarwereequal

numbersofAHLReceiver/CsgA,aTcReceiver/CsgAHis,AHLReceiver/GFP,andaTcReceiver/mCherry

cells.b,Bycombiningsyntheticgeneregulationwithspatialinducergradients,wecreated

achangeinthenanoscalestructureoffibrilsacrossadistanceofmillimetres.This

27


nanoscaleandmacroscalepatterningwasshownbychangesinsegmentlengthsof

unlabelledandNiNTA‐AuNP‐labelledfibrilsegmentsatdifferentlocationsacrosstheagar

plate.InducerconcentrationgradientsweredemonstratedbyoverlaidGFPandmCherry

fluorescenceimagesofembeddedAHLReceiver/GFPandandaTcReceiver/mCherryreporter

cells.Scalebarsare200nm.c,Wealsoachievedpatterningatthenanoscalebyprotein

engineeringofcurlisubunits.ConcatenatingeighttandemrepeatsofCsgAandaddingone

histidinetagtotheC‐terminus(8XCsgAHis)resultedinfibrilsthatwerelabelledbya

syncopatedpatternofNiNTA‐AuNPs,withclustersofparticlesseparatedby33.3±27.1

(s.e.m.)nm.Scalebarsare100nm.d,Syntheticgenecircuitsthatcouplecurlisubunit

secretiontoexternalinducersignals,whencombinedwithsubunitengineering,enable

patterningacrossmultiplelengthscales(nanometrestomicrometres).WeusedAHLto

induceproductionof8XCsgAHisfromAHLReceiver/8XCsgAHisandthenusedaTctoinduce

productionofCsgAHisfromaTcReceiver/CsgAHis.IntheTEMimages,dashedbrownlinesrefer

tosyncopated8XCsgAHissegmentswhilethesolidamethystlinesindicateCsgAHissegments.

DetailedhistogramsfordatashownherecanbefoundinSupplementaryFigure11.Scale

barsare100nm.

Interfaceswithinorganicmaterials

Ourlivingcellsystemcanbeusedtocreatefunctionalmaterials,suchas

environmentallyswitchableconductivebiofilms.WehypothesizedthataTc‐inducible

28

productionofCsgAHismonomersbyaTcReceiver/CsgAHiscellswouldgenerateextracellular

amyloidfibrilsthatorganizeNiNTA‐AuNPsintochainsandformaconductivebiofilm

network.AsshowninFigure1,theexpressionofextracellularcurlifibrilsenablessurface

adherencebymulticellularbacterialcommunities,resultinginbiofilmformation.

EngineeredbiofilmsweregrownoninterdigitatedelectrodesdepositedonThermanox

coverslips,withaTcReceiver/CsgAHiscellsculturedinthepresenceofNiNTA‐AuNPsandinthe

presenceorabsenceofaTcinducer(Fig.5a).Weshowedbyconfocalmicroscopythat

biofilmswereformedinanaTc‐dependentmanner(SupplementaryFig.14).Scanning

electronmicroscopy(SEM),scanningelectronmicroscopy/energydispersiveX‐ray

spectroscopy(SEM/EDS),andtransmissionelectronmicroscopy(TEM)wereperformedto

furthercharacterizebiofilmsamples(Fig.5b).InthepresenceofaTc,biofilmsformed,

spannedelectrodes(asshownbySEMimaging),andcontainednetworksofgoldthat

connectedelectrodes(asshownbySEM/EDSelementalmapping).Incontrast,SEM

imagingofcellsgrownintheabsenceofinductionshowedonlyscatteredbacteriainthe

gapsbetweenelectrodes,andSEM/EDSshowednogoldnetworks.TEMimagingrevealed

thataTc‐inducedbiofilmsorganizedgoldparticlesintodensenetworks(Fig.5band

SupplementaryFig.16),whilesampleswithcellsintheabsenceofaTcshowedonly

scattered,isolatedgoldparticles(Fig.5b).BiofilmsformedinthepresenceofaTchad

0.82±0.17(s.e.m.)nanosiemensconductance,whereassampleswithcellsintheabsenceof

aTchadnomeasureableconductance(SupplementaryFig.15).Biofilmsformedwith

aTcReceiver/CsgAHiscellsinducedbyaTc,butgrownintheabsenceofNiNTA‐AuNPs,had

electricalconductancethatwastwoordersofmagnitudelowerthanthoseformedin

presenceofNiNTA‐AuNPs(SupplementaryFig.17a).Samplescontaining

29

AHLReceiver/CsgAHiscellsgrowninthepresenceofNiNTA‐AuNPsandaTchadno

measureableconductance(SupplementaryFig.17b).

Weextendedcell‐basedgold‐particlepatterningtocreatenanowiresandnanorods

viaadditionalgolddeposition.WhenaTcReceiver/CsgAHisandAHLReceiver/CsgAcellswere

inducedwithonlyaTc,theresultingcurlifibrilstemplatedgoldnanowires.Whenthecells

wereinducedwithbothaTcandAHL,theresultingco‐fibrilscontainedCsgAHisandCsgA

whichtemplatedconsecutivegoldnanorods(Fig.5c).Goldnanorodshavebeenstudiedfor

arangeofapplicationsbecauseoftheirmorebroadlytunableabsorptionspectracompared

tonanoparticles,whichallowsforpeakabsorptioninthenear‐IRwindowusedforinvivo

imagingandphotothermalablation27.Moreover,viaconjugationwithtargetingligandsand

drugmolecules,theycanalsoactastargeteddrugdeliveryvehiclesfortherapeuticand

diagnosticapplications28,29.

30

Figure2‐5|Environmentallyswitchableconductivebiofilmsandcell‐based

synthesisofcurli‐templatednanowiresandnanorods.a,WeusedaTcReceiver/CsgAHis

cellstoformamyloidfibrilscomposedofCsgAHisinresponsetoaTc.Whencombinedwith

NiNTA‐AuNPs,wecreatedconductivebiofilmsthatcanbeexternallycontrolledas

electricalswitches.WhenaTcwasaddedtoaTcReceiver/CsgAHiscellsgrowninthepresence

ofNiNTA‐AuNPs,ittriggeredtheformationofconductivebiofilmsonelectrodes,with

embedded5nmgoldparticlesgivingbiofilmsaredcolour(‘ON’,solidredbox).However,in

theabsenceofaTc,fewcellsadheredtotheelectrodes(‘OFF’,dashedgreybox).Scalebars

are5mm.b,SEM/EDSelementalmappingoftheaTc‐induced‘ON’statefor

aTcReceiver/CsgAHisbiofilmsshowedthatnetworksofgoldinthebiofilmsconnectedthe

electrodes(whitearrows).SEMimagingshowedthatthebiofilmsbridgedelectrodes.TEM

imagingshowednetworksofaggregatedgoldparticles.Incontrast,SEM/EDSmappingof

31


the‘OFF’stateshowednogoldnetworks,SEMimagingshowedonlyscatteredcellsinthe

gapbetweenelectrodes,andTEMimagingshowedonlyscatteredandisolatedgold

particles(blackarrow).Scalebarsofscanningelectronmicrographsare20 ・ mandscale

barsoftransmissionelectronmicrographsare200nm.c,Amixedpopulationof

aTcReceiver/CsgAHisandAHLReceiver/CsgAcellsproducedcurlitemplatesfororganizingeither

goldnanowiresorgoldnanorodswhentheywereinducedwithaTconlyorbothaTcand

AHL,respectively.NiNTA‐AuNPswerepatternedonCsgAHissubunitswithincurlifibrilsand

thengoldenhanced.Scalebarsare200nm.

Wealsousedcellularbiofabricationtocreateco‐fibrilsthatassembledCdTe/CdS

quantumdots(QDs)withgoldnanoparticles,resultinginthemodulationofQD

fluorescence(Fig.6).WeleveragedinteractionsbetweentheSpyCatcherproteinandthe

SpyTagpeptidetag30,whichresultsintheformationofcovalentbonds,todockQDsto

fibrilsdisplayingSpyTag.Weusedanorthogonalinteractionbetweenanti‐FLAGantibodies

andtheFLAGaffinitytagtodock40nmgoldparticlestofibrilsdisplayingtheFLAGtag.

CsgASpyTagfibrilswerespecificallyboundbySpyCatcher‐conjugatedCdTe/CdSQDs(QD‐

SpyCatcher,SupplementaryFig.21),whileCsgAFLAGfibrilswerespecificallyboundbyanti‐

FLAGantibodieswhichwereinturnboundby40nmgoldparticlesconjugatedwith

secondaryantibodies(Fig.6a).AHLReceiver/CsgASpyTagandaTcReceiver/CsgAFLAGstrainswere

co‐culturedinthepresenceofAHL,aTc,orbothAHLandaTc.InthepresenceofAHL,fibrils

32

producedbythecellularconsortiaonlyboundQD‐SpyCatcher,whereasinthepresenceof

aTc,theresultingfibrilsonlyboundantibody‐conjugated40nmgoldparticles(Fig.6band

SupplementaryFig.18).Whenbothinducerswerepresent,thefibrilsco‐assembledQDs

withgoldnanoparticles(Fig.6b).Characterizationwithfluorescence‐lifetimeimaging

microscopy(FLIM)revealedthatco‐assembliesofQDsandgoldnanoparticleshadaltered

fluorescencelifetimesandintensitiescomparedtoassembliesofQDsalone(Fig.6c).

Theseresultsdemonstratethatthebehaviourofstimuli‐responsivematerialscanbe

modulatedbycurlifibrilspatternedwithengineeredcells.AuNP‐QDheterostructuresare

ofinterestbecauseplasmon‐excitoninteractionsbetweenplasmonicAuNPsand

fluorescentQDsallowfortailoringofphotonemissionproperties.Byselectingappropriate

materialsandarchitectures,onecanpotentiallytuneemissionintensity,directionality,and

spectralprofileforarangeofapplications31‐34.

Inadditiontoorganizingpre‐formednanomaterials,cell‐fabricatedcurlifibrilscan

beusedtogrowinorganicmaterials.Todemonstratethis,weengineeredastrainthat

producedcurlifibrilsdisplayingaZnS‐nucleatingpeptide(CsgAZnSpeptide,Supplementary

Table1)35.Theresultingfibrilsnucleated~5nmparticles(Fig.6d),whereascontrolfibrils

composedofwild‐typeCsgAnucleatedfewsuchparticles(Fig.6e).HRTEMimagesrevealed

thatthenucleatedparticleshadacubiczincblendeZnS(111)structurewithatypical

crystallinespacingof0.31nm(Fig.6d).EDSanalysisofelementalcompositionshowedan

approximately1:1ratioofzincandsulphur(Fig.6d).Thesedataindicatethattheparticles

areZnSnanocrystals.Thenanocrystalswerefluorescent,withanemissionpeakat490nm

whenexcitedat405nm(Fig.6d).

33

Figure2‐6|AssemblyandtuningoffunctionalAuNP‐QDheterostructuresand

nucleationoffluorescentZnSQDsoncell‐synthesizedcurlifibrils.a,CsgASpyTagfibrils

specificallybindCdTe/CdSQDsconjugatedtotheSpyCatcherprotein;theCdTecoresof

QDsareseenunderHRTEM.CsgAFLAGfibrilsarespecificallyboundbyanti‐FLAGantibodies

whichareinturnboundby40nmAuNPsconjugatedtosecondaryantibodies.b,Amixed

populationofaTcReceiver/CsgAFLAGandAHLReceiver/CsgASpyTagcellsproducedcurlitemplates

foreitherAuNP‐QDheterostructures(co‐fibrilsofCsgAFLAGandCsgASpyTag)orQD‐only

assemblies(CsgASpyTagfibrils)dependingonwhethertheywereinducedbybothaTcand

AHL,orAHLonly,respectively.c,Cell‐patternedcurlifibrilsenablethetuningofstimuli‐

responsiveinorganic‐organicmaterials.AuNP‐QDassembliespatternedon

CsgAFLAG/CsgASpyTagscaffolds(solidredbars)exhibiteddifferentfluorescencelifetimeand

intensitypropertiesthanQD‐onlyassembliespatternedonCsgASpyTagscaffolds(hashed

bluebars).d,CsgAZnSpeptidefibrilsnucleated~5nmnanoparticleswithacubiczincblende

ZnS(111)structureandapproximately1:1ratioofzincandsulphur.Theparticleswere

34


fluorescent,withanemissionpeakat490nmwhenexcitedat405nm.e,ControlCsgAfibrils

nucleatedfewsuchparticles.Ina),b),d),ande)blackscalebarsare200nmandwhitescale

barsare5nm;theimagesoutlinedbyredboxesarezoomed‐inversionsoftheinsetred

boxes.

Outlook

Wehaveshownthatprotein‐basedamyloidfibrilsproducedbylivingcellscanbe

interfacedwithdifferentinorganicmaterialsviaarangeofstrategies.Beyondbeinga

convenientmodelsystemwithwhichtoexploretheapplicationsoflivingsystemsto

materialsscience,proteinmaterialsareofpracticalinterestbecausetheyconstitutea

majorclassofbiomaterials36.Proteinmaterialscanhaveprogrammablestructures37and

diversefunctionalities,suchasresponsivenesstophysicochemicalstimuli38,theabilityto

interactwithlivingsystems39,andtheabilitytoorganizeabioticmaterialsforexpanded

functionalities35,40‐42.Amyloidfibrilscanprovidebeneficialmaterialspropertiessuchas

resistancetodegradationandmechanicalstrengthcomparabletothatofsteel43.Aswe

haveshownhere,amyloidfibrilsassembledbycellsconstituteaversatilescaffoldthatcan

co‐organizeandsynthesizefluorescentQDsaswellasgoldnanowires,nanorods,and

nanoparticles.ThisapproachcouldbegeneralizedtoincludemultipleCsgAvariantswith

differentfunctionalpropertiesandabilitiestointeractwithvariousinorganicmaterials.In

addition,curlifibrilswithtunablestructureandcompositioncouldbeusedaspatterned

35

scaffoldsformulti‐enzymesystemsbydisplayingorthogonalaffinitytagsoncurlifibers

whichinteractwithdifferentenzymes.

Mostexistingexamplesofproteinbiomaterialsareassembledinvitrofrom

chemicallysynthesizedpeptidesorpurifiedsubunitsanddonottakefulladvantageofthe

factthatthematerials’constituentsubunitscanbeintegratedintolivingcellcommunities.

Livingcellsarenaturalplatformsforengineeringmultiscalepatternedmaterialsbecause

biologyisorganizedinahierarchicalmanner,frommacromolecules(e.g.proteins,nucleic

acids,carbohydrates,lipids)tomacromolecularassemblies(syntheticvariantsofwhichare

usedasnanomaterials37,44‐46)toorganellestocellsandtotissues.Infact,naturalbiological

materialssuchasbonearehierarchicallyorganizedtofulfilvariedfunctional

requirements1,9.Thus,wehavedemonstratedanengineeredcellularplatformthat

synthesizesandpatternsself‐assemblingmaterialswithcontrollablefunctionality,

structure,andcomposition.

Usinggenecircuitswithinengineeredbiofilmsformultiscalepatterningofmaterials

isanovelapplicationareaforsyntheticbiology.Thisworkappliesusefulcharacteristicsof

multicellularcommunitiestomaterialsfabricationandbuildsuponpreviouseffortsto

engineerbiofilmswithsyntheticcircuits10‐16.Thisstrategycanbeexpandedtoother

cellularandbiomaterialscontextsforapplicationsrangingfrombiointegratedelectronic

andopticaldevices47‐49totissueengineeringscaffolds50.Forexample,cellsdesignedto

computeandintegratecomplexsignalscouldbeusedtoassemblefunctionalmaterialsin

responsetotheirenvironment18,19.These“smart”livingmaterialscouldbecomposedof

specializedcellularconsortiathatcoordinatewitheachotherformulti‐functionalmaterials

36

synthesis.Mammaliancellscapableoftunable,environmentallyresponsivesynthesisof

multiscalematerialscouldbeusedtomimicthedynamicmicroenvironmentofinvivo

extracellularmatrices51fortissueengineering.Ourdemonstrationofagradientmaterial

patternedatthenanoscaleandthemillimetrescalecouldbeusedtobiofabricate

functionallygradedmaterials52.Moreover,leveraginghierarchicalorganizationfrom

biologyformultiscalepatterningshouldcomplementotherstrategiesformaterials

synthesisthatrequiredirectedintervention53,suchas3Dprinting52.Repeatedmaterials‐

synthesisprocessesorenvironmentallyswitchablebehaviourscouldbeachievedby

triggeringbiofilmdisassembly12,13,54.

Insummary,byintegratingsyntheticgenenetworksinengineeredcellswith

extracellularproteinbiomaterials,livingmaterialswithenvironmentalresponsiveness,

tunablefunctionalities,multiscalepatterning,andeventheabilitytoself‐healandremodel

couldberealized.Insuchmaterials,therewouldbeadivisionoflabourbetweencells

(providingfunctionalitiesoflivingsystems)55,extracellularproteinmaterials(providing

spatialpatterningandstructuralintegrity),andinterfacedabioticmaterials(providing

functionalitiesofnon‐livingsystems).Thus,weenvisionthatengineeringartificialcellular

consortia,suchasbiofilms,tosynthesizeandorganizeheterogeneousfunctionalmaterials

willenabletherealizationofsmartcompositematerialsthatcombinethepropertiesof

livingandnon‐livingsystems.

37

MethodsSummary

Cultureconditions.SeedcultureswereinoculatedfromglycerolstocksandgrowninLB‐

Millermediumfor12hat37°C.Experimentalculturesweregrownat30°CinM63minimal

mediumsupplementedwith1mMMgSO4andwith0.2%w/vglucoseor0.2%w/vglycerol.

Forinducingconditions,anhydrotetracycline(Sigma)atconcentrationsof1‐250ng/mland

N‐(β‐ketocaproyl)‐L‐homoserinelactone(Sigma)atconcentrationsof1‐1000nMwere

used.

Anti‐CsgAimmuno‐labelling.Rabbitanti‐CsgAprimaryantibody(M.Chapman,University

ofMichigan)wasusedat1:1000dilution,goatanti‐rabbitsecondaryantibodyconjugated

to10nmgoldparticles(Sigma)wasusedat1:10dilution.

NiNTA‐AuNPlabelling.ForspecificbindingofNiNTA‐AuNP(Nanoprobes)tohistidine

tagsdisplayedoncurlifibrils,bufferconsistingof1XPBSwith0.487MNaCl,80mM

imidazole,and0.2v/v%Tween20wasused.

Conductivebiofilmconductancemeasurement.Interdigitatedelectrodes(IDEs)for

measuringbiofilmconductancewerecreatedbysputteringgoldthroughcustom

shadowingmasks(Tech‐Etch)ontoThermanoxcoverslips(Nunc).IDEswereplacedin24‐

wellplatewellsandconductivebiofilmsgrownbyadding100nMNiNTA‐AuNPintoculture

medium.Afterbiofilmculture,IDEswerewashedbyrepeatedlyimmersinginddH2O,laid

onaflatsurface,andallowedtoairdryforthreedays.AKeithley4200picoammeterwith

two‐pointprobewasusedtocarryoutavoltagesweep.

38

Goldnanowireandnanorodsynthesis.GoldwasspecificallydepositedonNiNTA‐AuNP

chainsusingGoldEnhance™EMkit(Nanoprobes).

SpecificbindingofQD‐SpyCatcher.ForspecificbindingofCdTe/CdS‐SpyCatcherto

SpyTagpeptidetagsdisplayedoncurlifibrils,bufferconsistingof1XPBS+350mMNaCl+

0.3v/v%Tween20wasused.

Specificbindingofantibody‐conjugated40nmAuNP.Rabbitanti‐FLAGprimary

antibody(Sigma)wasusedat1:250dilution,goatanti‐rabbitsecondaryantibody

conjugatedto40nmAuNPs(Abcam)wasusedat1:10dilution.

Zincsulphidenanocrystalsynthesis.Fibrilsampleswereincubatedwith1µMZnCl2at

RTfor12h,followedbyadditionof1µMNa2S.Sampleswerethenincubatedat0°Cfor24h

bypackinginiceandplacingina4°Croom,andsubsequentlyallowedtoagefor12hatRT.

Transmissionelectronmicroscopy.Samplesweredepositedon200‐mesh

formvar/carboncoatednickelTEMgridsandstainedwith2%uranylacetate.TEMimages

wereobtainedonaFEITecnaiSpirittransmissionelectronmicroscopeoperatedat80kV

acceleratingvoltage.High‐resolutiontransmissionelectronmicroscopy(HRTEM)and

energy‐dispersiveX‐rayspectroscopy(EDS)wereperformedonaJEOL2010Felectron

microscopeoperatingat200kV.

Scanningelectronmicroscopy.SampleswereimagedwithaJEOLJSM‐6010LAscanning

electronmicroscopeoperatedat10kVacceleratingvoltage.Imageswereobtainedin

secondaryelectronimaging(SEI)mode,andelementalmappingwasperformedwith

energy‐dispersiveX‐rayspectroscopy(EDS).

39

Fluorescencemicroscopy.Fluorescence‐lifetimeimagingmicroscopy(FLIM)was

performedwithaZeiss710NLOmultiphotonmicroscopewith20Xobjectiveand

connectedtoatime‐correlatedsingle‐photoncountingsystem(Becker&Hickl).The

excitationsourcewasa2‐photonlaser(CoherentChameleonVisionII)tunedto800nm,

andemissionwasdetectedthrougha590‐650nmbandpassfilter.Lambdascananalysisof

fluorescentZnSnanocrystalswasperformedwithaZeissLSM710NLOLaserScanning

Confocalwith10Xobjectiveand405nmexcitationlaser.

40

References

1 Fratzl,P.&Weinkamer,R.Nature’shierarchicalmaterials.ProgressinMaterialsScience52,1263‐1334,doi:http://dx.doi.org/10.1016/j.pmatsci.2007.06.001(2007).

2 Kollmannsberger,P.,Bidan,C.M.,Dunlop,J.W.C.&Fratzl,P.Thephysicsoftissue

patterningandextracellularmatrixorganisation:howcellsjoinforces.SoftMatter7,9549‐9560,doi:10.1039/c1sm05588g(2011).

3 Stevens,M.M.&George,J.H.Exploringandengineeringthecellsurfaceinterface.

Science310,1135‐1138,doi:10.1126/science.1106587(2005).4 O'Toole,G.,Kaplan,H.B.&Kolter,R.Biofilmformationasmicrobialdevelopment.

AnnuRevMicrobiol54,49‐79,doi:10.1146/annurev.micro.54.1.4954/1/49[pii](2000).

5 Epstein,A.K.,Pokroy,B.,Seminara,A.&Aizenberg,J.Bacterialbiofilmshowspersistentresistancetoliquidwettingandgaspenetration.ProcNatlAcadSciUSA108,995‐1000,doi:1011033108[pii]10.1073/pnas.1011033108(2011).

6 Belcher,A.M.etal.Controlofcrystalphaseswitchingandorientationbysolublemollusc‐shellproteins.Nature381,56‐58(1996).

7 Su,X.W.,Zhang,D.M.&Heuer,A.H.TissueRegenerationintheShelloftheGiant

QueenConch,Strombusgigas.Chem.Mater.16,581‐593(2004).8 Aizenberg,J.etal.SkeletonofEuplectellasp.:structuralhierarchyfromthe

nanoscaletothemacroscale.Science309,275‐278,doi:10.1126/science.1112255(2005).

9 Weiner,S.&Wagner,H.D.Thematerialbone:Structuremechanicalfunction

relations.Annu.Rev.Mater.Sci.28,271‐298,doi:10.1146/annurev.matsci.28.1.271(1998).

10 Brenner,K.&Arnold,F.H.Self‐organization,layeredstructure,andaggregation

enhancepersistenceofasyntheticbiofilmconsortium.PloSone6,e16791,doi:10.1371/journal.pone.0016791(2011).

11 Brenner,K.,Karig,D.K.,Weiss,R.&Arnold,F.H.Engineeredbidirectional

communicationmediatesaconsensusinamicrobialbiofilmconsortium.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica104,17300‐17304,doi:10.1073/pnas.0704256104(2007).

41

12 Hong,S.H.etal.Syntheticquorum‐sensingcircuittocontrolconsortialbiofilmformationanddispersalinamicrofluidicdevice.Naturecommunications3,613,doi:10.1038/ncomms1616(2012).

13 Ma,Q.,Yang,Z.,Pu,M.,Peti,W.&Wood,T.K.Engineeringanovelc‐di‐GMP‐binding

proteinforbiofilmdispersal.Environmentalmicrobiology13,631‐642,doi:10.1111/j.1462‐2920.2010.02368.x(2011).

14 Lee,J.,Jayaraman,A.&Wood,T.K.Indoleisaninter‐speciesbiofilmsignalmediated

bySdiA.BMCmicrobiology7,42,doi:10.1186/1471‐2180‐7‐42(2007).15 Payne,S.etal.Temporalcontrolofself‐organizedpatternformationwithout

morphogengradientsinbacteria.Molecularsystemsbiology9,697,doi:10.1038/msb.2013.55(2013).

16 Payne,S.&You,L.EngineeredCell‐CellCommunicationandItsApplications.

Advancesinbiochemicalengineering/biotechnology,doi:10.1007/10_2013_249(2013).

17 Barnhart,M.M.&Chapman,M.R.Curlibiogenesisandfunction.AnnuRevMicrobiol

60,131‐147,doi:10.1146/annurev.micro.60.080805.142106(2006).18 Callura,J.M.,Cantor,C.R.&Collins,J.J.Geneticswitchboardforsyntheticbiology

applications.ProcNatlAcadSciUSA109,5850‐5855,doi:10.1073/pnas.1203808109(2012).

19 Prigent‐Combaret,C.etal.Developmentalpathwayforbiofilmformationincurli‐

producingEscherichiacolistrains:roleofflagella,curliandcolanicacid.Environmentalmicrobiology2,450‐464(2000).

20 Vidal,O.etal.IsolationofanEscherichiacoliK‐12mutantstrainabletoform

biofilmsoninertsurfaces:involvementofanewompRallelethatincreasescurliexpression.Journalofbacteriology180,2442‐2449(1998).

21 Hung,C.etal.Escherichiacolibiofilmshaveanorganizedandcomplexextracellular

matrixstructure.mBio4,e00645‐00613,doi:10.1128/mBio.00645‐13(2013).22 Wang,X.,Hammer,N.D.&Chapman,M.R.Themolecularbasisoffunctional

bacterialamyloidpolymerizationandnucleation.TheJournalofbiologicalchemistry283,21530‐21539,doi:10.1074/jbc.M800466200(2008).

23 Basu,S.,Gerchman,Y.,Collins,C.H.,Arnold,F.H.&Weiss,R.Asynthetic

multicellularsystemforprogrammedpatternformation.Nature434,1130‐1134,doi:nature03461[pii]10.1038/nature03461(2005).

42

24 BacchusW,L.M.,El‐BabaMD,WeberW,StellingJ,FusseneggerM.Synthetictwo‐waycommunicationbetweenmammaliancells.NatBiotechnol.30,991‐996(2012).

25 Tabor,J.J.etal.Asyntheticgeneticedgedetectionprogram.Cell137,1272‐1281,

doi:S0092‐8674(09)00509‐1[pii]10.1016/j.cell.2009.04.048(2009).

26 Liu,C.etal.Sequentialestablishmentofstripepatternsinanexpandingcellpopulation.Science334,238‐241,doi:10.1126/science.1209042(2011).

27 Jang,B.,Park,J.Y.,Tung,C.H.,Kim,I.H.&Choi,Y.Goldnanorod‐photosensitizer

complexfornear‐infraredfluorescenceimagingandphotodynamic/photothermaltherapyinvivo.ACSnano5,1086‐1094,doi:10.1021/nn102722z(2011).

28 Dreaden,E.C.etal.Smallmolecule‐goldnanorodconjugatesselectivelytargetand

inducemacrophagecytotoxicitytowardsbreastcancercells.Small8,2819‐2822,doi:10.1002/smll.201200333(2012).

29 Libutti,S.K.etal.PhaseIandpharmacokineticstudiesofCYT‐6091,anovel

PEGylatedcolloidalgold‐rhTNFnanomedicine.Clinicalcancerresearch:anofficialjournaloftheAmericanAssociationforCancerResearch16,6139‐6149,doi:10.1158/1078‐0432.CCR‐10‐0978(2010).

30 Zakeri,B.etal.Peptidetagformingarapidcovalentbondtoaprotein,through

engineeringabacterialadhesin.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica109,E690‐697,doi:10.1073/pnas.1115485109(2012).

31 Polman,A.&Atwater,H.A.Photonicdesignprinciplesforultrahigh‐efficiency

photovoltaics.NatMater11,174‐177,doi:Doi10.1038/Nmat3263(2012).32 Reineck,P.etal.ASolid‐StatePlasmonicSolarCellviaMetalNanoparticleSelf‐

Assembly.AdvMater24,4750‐4755,doi:DOI10.1002/adma.201200994(2012).33 Curto,A.G.etal.UnidirectionalEmissionofaQuantumDotCoupledtoa

Nanoantenna.Science329,930‐933,doi:DOI10.1126/science.1191922(2010).34 Yuan,Z.L.etal.Electricallydrivensingle‐photonsource.Science295,102‐105,

doi:DOI10.1126/science.1066790(2002).35 Mao,C.etal.Viralassemblyoforientedquantumdotnanowires.Proceedingsofthe

NationalAcademyofSciencesoftheUnitedStatesofAmerica100,6946‐6951,doi:10.1073/pnas.0832310100(2003).

36 Zhang,S.G.Fabricationofnovelbiomaterialsthroughmolecularself‐assembly.

Naturebiotechnology21,1171‐1178,doi:10.1038/nbt874(2003).

43

37 King,N.P.etal.Computationaldesignofself‐assemblingproteinnanomaterialswithatomiclevelaccuracy.Science336,1171‐1174,doi:10.1126/science.1219364(2012).

38 Mart,R.J.,Osborne,R.D.,Stevens,M.M.&Ulijn,R.V.Peptide‐basedstimuli‐

responsivebiomaterials.SoftMatter2,822‐835,doi:10.1039/b607706d(2006).39 Webber,M.J.etal.SupramolecularnanostructuresthatmimicVEGFasastrategyfor

ischemictissuerepair.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica108,13438‐13443,doi:10.1073/pnas.1016546108(2011).

40 So,C.R.,Tamerler,C.&Sarikaya,M.Adsorption,diffusion,andself‐assemblyofan

engineeredgold‐bindingpeptideonAu(111)investigatedbyatomicforcemicroscopy.AngewChemIntEdEngl48,5174‐5177,doi:10.1002/anie.200805259(2009).

41 Channon,K.J.,Devlin,G.L.&MacPhee,C.E.Efficientenergytransferwithinself‐

assemblingpeptidefibers:aroutetolight‐harvestingnanomaterials.JournaloftheAmericanChemicalSociety131,12520‐12521,doi:10.1021/ja902825j(2009).

42 Scheibel,T.etal.Conductingnanowiresbuiltbycontrolledself‐assemblyofamyloid

fibersandselectivemetaldeposition.ProcNatlAcadSciUSA100,4527‐4532,doi:10.1073/pnas.04310811000431081100[pii](2003).

43 Smith,J.F.,Knowles,T.P.,Dobson,C.M.,Macphee,C.E.&Welland,M.E.Characterizationofthenanoscalepropertiesofindividualamyloidfibrils.ProcNatlAcadSciUSA103,15806‐15811,doi:0604035103[pii]10.1073/pnas.0604035103(2006).

44 Felgner,P.L.etal.Lipofection:ahighlyefficient,lipid‐mediatedDNA‐transfectionprocedure.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica84,7413‐7417(1987).

45 Winfree,E.,Liu,F.,Wenzler,L.A.&Seeman,N.C.Designandself‐assemblyoftwo‐

dimensionalDNAcrystals.Nature394,539‐544,doi:10.1038/28998(1998).46 Rothemund,P.W.FoldingDNAtocreatenanoscaleshapesandpatterns.Nature

440,297‐302,doi:10.1038/nature04586(2006).47 Tian,B.etal.Macroporousnanowirenanoelectronicscaffoldsforsynthetictissues.

Naturematerials11,986‐994,doi:10.1038/nmat3404(2012).48 Hwang,S.W.etal.Aphysicallytransientformofsiliconelectronics.Science337,

1640‐1644,doi:10.1126/science.1226325(2012).

44

49 Amsden,J.J.etal.Rapidnanoimprintingofsilkfibroinfilmsforbiophotonicapplications.AdvMater22,1746‐1749,doi:10.1002/adma.200903166(2010).

50 Lutolf,M.P.&Hubbell,J.A.Syntheticbiomaterialsasinstructiveextracellular

microenvironmentsformorphogenesisintissueengineering.Naturebiotechnology23,47‐55,doi:10.1038/nbt1055(2005).

51 Prewitz,M.C.etal.Tightlyanchoredtissue‐mimeticmatricesasinstructivestemcell

microenvironments.Naturemethods10,788‐794,doi:10.1038/nmeth.2523(2013).52 Chiu,W.K.&Yu,K.M.Directdigitalmanufacturingofthree‐dimensional

functionallygradedmaterialobjects.Computer‐AidedDesign40,1080–1093(2008).53 Xia,Y.,Rogers,J.A.,Paul,K.E.&Whitesides,G.M.UnconventionalMethodsfor

FabricatingandPatterningNanostructures.Chemicalreviews99,1823‐1848(1999).54 Kolodkin‐Gal,I.etal.D‐aminoacidstriggerbiofilmdisassembly.Science328,627‐

629,doi:10.1126/science.1188628(2010).55 Gubeli,R.J.,Burger,K.&Weber,W.Syntheticbiologyformammaliancelltechnology

andmaterialssciences.Biotechnologyadvances31,68‐78,doi:10.1016/j.biotechadv.2012.01.007(2013).

45

Appendix

46

SupplementaryFigures

SupplementaryFigureA‐1|Immuno‐labellingofcurlifibrilswithrabbitanti‐CsgA

antibodiesandgold‐conjugatedgoatanti‐rabbitantibodies.ThisdatashowsthatE.coli

MG1655ompR234cells(“ompR234”,seeSupplementaryTable3),whichhaveanintact

endogenouscsgAgene,producecurlifibrilsthatarelabelledbyanti‐CsgAantibodies

andattachedtocells.However,cellswiththecsgAgeneknockedoutandnocsgA‐

expressingcircuits(“csgAompR234”,seeSupplementaryTable3),and

aTcReceiver/CsgAHiscellsintheabsenceofaTc,didnotproducecurlifibrils.Inducing

aTcReceiver/CsgAHiscellswithaTcenabledtheproductionofcurlifibrilsthatwere

labelledbyanti‐CsgAantibodiesandattachedtocells.Thiswasthecaseforallculture

conditionsused(seeSupplementaryMethods):liquidM63withglucoseonThermanox

47

SupplementaryFigureA‐1(Continued)

(Fig.1b),liquidM63withglucoseonpolystyrene,liquidM63withglycerolon

polystyrene,andM63withglucosein0.7%agar.Scalebarsare200nm.

48

SupplementaryFigureA‐2|a,Confocalmicroscopyandbiomassquantification

revealedthatunderstaticcultureconditionswithliquidM63mediacontainingglycerol,

E.coliMG1655ompR234(“ompR234”),whichhasanintactendogenouscsgAgene,

formedthickadherentbiofilms.However,cellswiththecsgAgeneknockedoutandno

csgA‐expressingcircuits(“csgAompR234”),andaTcReceiver/CsgAHiscellsintheabsence

ofaTc,didnotformbiofilms.Incontrast,inducingaTcReceiver/CsgAHiscellswithaTc

resultedintheformationofthickadherentbiofilms.b,Confocalmicroscopyand

biomassquantificationrevealedsimilarbiofilm‐formingcapabilitiesbyMG1655

ompR234andinducedaTcReceiver/CsgAHiscellswhengrowninflowcellswithliquidM63

mediacontainingglycerol.Inthisfigure,a)andb)arecomplementaryexperimentsto

thoseshowninFig.1cand1d,whichusedliquidM63mediawithglucose;here,weused

liquidM63mediawithglycerol.Toenablevisualization,wetransformedaconstitutive

mCherry‐expressingplasmidintoallstrains(seeSupplementaryMethods).Orthogonal

XZandYZviewsaremaximum‐intensityprojections.Scalebarsare50µm.

49

SupplementaryFigureA‐3|CrystalViolet(CV)stainingrevealedthatunderstatic

cultureconditionswithliquidM63containingglucose,nobiofilmswereformedby

aTcReceiver/CsgAHiscellsintheabsenceofaTc,orbycellswiththecsgAgeneknockedout

(MG1655PROcsgAompR234).Incontrast,adherentbiofilmsareformedby

aTcReceiver/CsgAHiscellsinthepresenceofaTc,andbyE.coliMG1655ompR234.CV

stainingwasquantifiedbyabsorbanceat550nm.

50

SupplementaryFigureA‐4|QuantitativeCongoRed(CR)bindingassayofBW25113

csgAcells,cellsexpressingCsgA(BW25113 csgA/pZS3‐pL(lacO)‐csgA),andcells

expressingCsgAwithaHistagfusedtotheC‐terminus(BW25113 csgA/pZS3‐

pL(lacO)‐csgAHisafterC‐terminus)showsthatCsgAmaintainsabilitytoformamyloidfibrils

afterfusionofpeptidestotheC‐terminus.

51

SupplementaryFigureA‐5|FibrilsimagedwithTEMareCsgA‐basedfibrilsand

histidinetagadditiontoCsgAdoesnotinterferewithfibrilproduction.Histidinetags

conferspecificNiNTAgoldparticle(NiNTA‐AuNP)bindingfunctionality.Fibrils

producedbyengineeredcellstrainsgrownwithchemicalinduction(b‐g)havesimilar

morphologytoCsgA‐basedcurlifibrilsdescribedinChapmanetal.1aswellasa,curli

fibrilsproducedbyE.coliMG1655ompR234.b,TherewasnospecificlabellingofCsgA

fibrilswithNiNTA‐AuNPsintheabsenceofhistidinetagsonCsgA(fibrilsproducedby

inducedaTcReceiver/CsgAcells).c,CurlifibrilswerespecificallylabelledbyNiNTA‐AuNPs

whenhistidinetagswereaddedtoCsgA(fibrilsproducedbyinducedaTcReceiver/CsgAHis

cells).Furthermore,histidinetagadditiontoCsgAdidnotinterferewithcurlifibril

production.d,EngineeredcellsexpressingCsgAwheninducedbyAHL

(AHLReceiver/CsgA)producedfibrils,asdide,engineeredcellsexpressingCsgAHiswhen

52


inducedbyAHL(AHLReceiver/CsgAHis).f,EngineeredcellsexpressingCsgAwheninduced

byaTc(aTcReceiver/CsgA)producedfibrils,asdidg,engineeredcellsexpressingCsgAHis

wheninducedbyaTc(aTcReceiver/CsgAHis).Scalebarsare200nm.

53

SupplementaryFigureA‐6|TuningthelengthdistributionofCsgAandCsgAHisblocks

bychangingtemporalintervallengthsandamplitudesofinputsignals.a,Histogram

datacorrespondingtoTEMimagesinFig.2b.Thesegmentsinblockco‐fibrilswere

longerthanthoseinco‐fibrilsassembledwhenCsgAandCsgAHisweresecreted

54


simultaneouslywithnotemporalseparation(Control),eventhoughbothtypesof

materialshadsimilarratiosofCsgAtoCsgAHis.Scalebaris200nm.b,Histogramdata

correspondingtoTEMimagesinFig.2d.Hashedbluebarsindicatebaresegmentsof

amyloidfibrilsthatwereunlabelledbyNiNTA‐AuNPs,whilesolidredbarsindicate

amyloidfibrilsegmentslabelledwithNiNTA‐AuNPs.

55

SupplementaryFigureA‐7|Conversionofchemicalinduceramplitudetomaterial

structureandcomposition.ComplementaryexperimenttothatdescribedinFig.2d.

Withsyntheticgenecircuits,cellscantranslatetheamplitudeofinputsignalsinto

nanoscalestructureandcompositionofblockco‐fibrils.AHLinducessecretionofCsgA

fromAHLReceiver/CsgA,whileatthesametime,aTcinducessecretionofCsgAHisfrom

aTcReceiver/CsgAHis.WetunedthelengthdistributionsoftheCsgAandCsgAHisblocks,as

wellastherelativeproportionsofCsgAandCsgAHis,bychangingtherelative

concentrationofsimultaneouslyappliedAHLandaTcinducers.Hashedbluebars

indicatebaresegmentsofamyloidfibrilsthatwereunlabelled,whilesolidredbars

56


indicateamyloidfibrilsegmentslabelledwithNiNTA‐AuNPs.Solidgreylineindicates

theproportionoffibrillengthlabelledbyNiNTA‐AuNPs.Scalebarsare200nm.

SupplementaryFigureA‐8|EngineeredE.colionlyproducecurlifibrilswheninduced

bythecorrectinducer.Thecurliknockouthoststrainusedtocreateengineered

inducer‐responsivestrains,E.coliMG1655PROcsgAompR234(“csgAompR234”),

doesnotproducecurlifibrils.Incontrast,theE.coliMG1655ompR234(“ompR234”)

straindoes.TheAHL‐responsiveengineeredstrain(AHLReceiver/CsgAHis)onlyproduces

curlifibrilswheninducedbyAHL.TheaTc‐responsiveengineeredstrain

(aTcReceiver/CsgAHis)onlyproducescurlifibrilswheninducedbyaTc.Curlifibril

productionwasquantifiedbytakingtheratioofareaoffibrilstoareaofcellsinTEM

imagesanalysedbyImageJ.

57

SupplementaryFigureA‐9|TheinteractionbetweenNiNTA‐AuNPsandHis‐tagged

curlifibrilsisspecific.His‐taggedCsgAfibrils(CsgAHis)boundNiNTA‐AuNPsbutwild‐

typeCsgAfibrilsdidnot,indicatingthattheHistagwasnecessaryforthisinteraction.

Moreover,goldparticleswithoutNiNTAconjugation(AuNP)didnotbindHis‐tagged

CsgAfibrils,indicatingthatNiNTAgroupswerealsorequiredforthisinteraction.The

imagesoutlinedbythickredboxesarezoomed‐inversionsofregionsoutlinedbythe

smallerredboxes.Scalebarsare200nm.

58

SupplementaryFigureA‐10|HistogramdatacorrespondingtotheTEMimagesinFig.

3b.ThisdatashowsthatthelengthdistributionofCsgAandCsgAHisblockschangedwith

timewhencurlisynthesiswasregulatedbyautonomouscellularcommunication

circuits.Hashedbluebarsindicatebaresegmentsofamyloidfibrilsthatwereunlabelled

byNiNTA‐AuNPs,whilesolidredbarsindicateamyloidfibrilsegmentslabelledwith

NiNTA‐AuNPs.

59

SupplementaryFigureA‐11|a,HistogramdatacorrespondingtoTEMimagesinFig.

4b.ThisdatashowsthatthelengthdistributionofCsgAandCsgAHisblockschanged

acrosstheagarplatewheninducerconcentrationgradientswerepresent,butnotinthe

absenceofgradients.Hashedbluebarsindicatebaresegmentsofamyloidfibrilsthat

wereunlabelledbyNiNTA‐AuNPs,whilesolidredbarsindicateamyloidfibrilsegments

labelledwithNiNTA‐AuNPs.b,HistogramdatacorrespondingtoTEMimagesinFig.4c.

60


ThisdatashowsthatCsgAHisand8XCsgAHisfibrilsorganizedNiNTA‐AuNPsindifferent

distributions.Hashedbluebarsindicatebaresegmentsofamyloidfibrilsthatwere

unlabelledbyNiNTA‐AuNPs,whilesolidredbarsindicateamyloidfibrilsegments

labelledwithNiNTA‐AuNPs.c,HistogramdatacorrespondingtoTEMimagesinFig.4d.

Thelengthdistributionsof8XCsgAHissegmentsandCsgAHissegmentsareshownin

histogramswithhashedbrownbarsandsolidamethystbars,respectively.Inset

histogramsshowthelengthdistributionsofunlabelledsegments(hashedbluebars)

andsegmentslabelledwithNiNTA‐AuNPs(solidredbars)forboththe8XCsgAHis

segmentsandtheCsgAHissegments.

61

SupplementaryFigureA‐12|Inducergradientvisualizationforagarplatesdescribed

inFig.4aandFig.4b.Top‐agar‐embeddedAHLReceiver/GFPreportercells(dashedgreen

lines)andaTcReceiver/mCherryreportercells(solidredlines)enabledthevisualizationof

inducerconcentrationgradientsacrosstheagarplate.

62

SupplementaryFigureA‐13|Shadowingmaskusedtomakeinterdigitatedelectrodes.

Thetwocontinuousyellowregionsrepresentholesinthemaskthroughwhichgoldwas

shadowedtocreatetheelectrodes.

63

SupplementaryFigureA‐14|a,Confocalmicroscopyandbiomassquantification

revealedthickbiofilmsformedbyaTc‐inducedaTcReceiver/CsgAHiscellsby24hours

whengrownwith100nMNiNTA‐AuNPinliquidM63mediacontainingglucose,under

staticcultureconditions.Toenablevisualization,wetransformedaconstitutive

mCherry‐expressingplasmidintothisstrain(seeSupplementaryMethods).b,Thesame

wasobservedunderflowconditions.Scalebarsare50µm.

64

SupplementaryFigureA‐15|TheaTc‐induced‘ON’stateforaTcReceiver/CsgAHis

biofilmsexhibitedaconductanceof0.82±0.17(s.e.m.)nS(solidredline),whilethe‘OFF’

statehadnomeasureableconductance(dashedgreyline).

65

SupplementaryFigureA‐16|TEMshowsthataTc‐inducedaTcReceiver/CsgAHiscells

producedcurlifibrilsthatorganizedNiNTA‐AuNPsintodensenetworks(the‘ON’state).

ThisiscomplementarydatatoTEMimagesshowinFig.5b.Theimageoutlinedbythe

thickredboxisazoomed‐inversionoftheregionoutlinedbythesmallredbox.Scale

baris1 m.

66

SupplementaryFigureA‐17|Additionalnegativecontrolsforchemical‐inducer‐

switchableconductivebiofilmsshowinFigure5.a,WhenaTcReceiver/CsgAHisbiofilms

weregrownwithaTcinductionbutwithoutNiNTA‐AuNPs,conductancewas2.4±3.5

(s.e.m.)pS.SEM/EDSshowednogoldnetworksconnectingelectrodes(whitearrows)

eventhoughSEMimagingshowedthatthickbiofilmsspannedelectrodes.TEMimaging

showedthatcurlifibrilswerepresent,butwithoutgoldparticles.b,Populationsofcells

whichdonotrespondtoaTc(AHLReceiver/CsgAHis)werenotconductiveinthepresence

ofaTcandNiNTA‐AuNPs.SEM/EDSshowednogoldnetworksconnectingelectrodes

andSEMimagingshowedonlyscatteredcellsinthegapbetweenelectrodes.TEM

67


imagingshowednocurlifibrilsandonlyscattered,isolatedgoldparticles.Scalebarsof

digitalphotographsare5mm,scalebarsofscanningelectronmicrographsare20 m,

andscalebarsoftransmissionelectronmicrographsare200nm.

68

SupplementaryFigureA‐18|AmixedpopulationofaTcReceiver/CsgAFLAGand

AHLReceiver/CsgASpyTagcellsproducedcurlitemplatesforAuNP‐onlyassemblies(CsgAFLAG

fibrils)wheninducedbyaTconly.FLAGtagsdisplayedonfibrilsspecificallyboundanti‐

FLAGantibodieswhichwereinturnboundbyAuNPsconjugatedtosecondary

antibodies.Theimageontherightisthezoomed‐inversionoftheinsetredboxonthe

left.Blackscalebarsare200nmandwhitescalebarsare5nm.

69

SupplementaryFigureA‐19|Syntheticriboregulatorsenabletightregulationofcurli

subunitexpression,whichisimportantforpatterning(Figs.2‐4).Whentheonlymeans

ofcontrolofcsgAgeneexpressionwasapL(lacO)promotercontrollingthedownstream

csgAgene(withnoriboregulators)incellsexpressingtheLacIrepressor(BW25113

ΔcsgA/pZS3‐pL(lacO)‐csgA+pZS4Int‐lacI/tetR),curlifibrilswereseenintheabsence

aswellasinthepresenceofIPTGinducer,makingsuchasystemnotsuitablefor

patterning.Scalebarsare200nm.

70

SupplementaryFigureA‐20|SequencingresultsofthePCRproductfromthecsgBAC

operonofE.coliMG1655PRO∆csgAompR234showthatcsgAwasdeletedandreplaced

byascarsequence,whilecsgBandcsgCareintact.Moreover,thepromoterforcsgBis

present.a,Thezoomed‐outsequencingresultsshowinganoverviewofthesequenced

operon.b,Thezoomed‐insequencingresultsshowingtheunderlyingDNAsequences.

Theconsensussequenceobtainedfromintegratingthreesequencingreactionsisthe

sequenceabovethegreenbar.Thesequencejustbelowthegreenbarrepresentsthe

referencesequence,whichisfurtherannotatedwithyellow,grey,andgreenarrows

representinggenes,FRTsites,andpromoterelements,respectively.Thegreenbar

belowtheconsensussequenceindicatesthatthesequencingresultsmatchthe

referencesequence.

a

b

71


72


73


74

SupplementaryFigureA‐21|Photoluminescencespectraandreal‐colorpicturesof

CdTe/CdSquantumdotsbefore(leftbluecurve,emissionpeakat620nm)andafter

(rightgreencurve,emissionpeakat638nm)conjugationwithCCSpyCatcherprotein

showthatCdTe/CdSQDsremainedhighlyfluorescentafterconjugation.

Photoluminescencespectrawereobtainedunderexcitationat450nm(NanoLog

Spectrometer,HORIBAJobinYvon).Real‐colorpictureswereobtainedunderexcitation

at365nm(UVlamp).

75

SupplementaryFigureA‐22|DotblotquantitationofCsgAproductioninbiofilms.We

quantitatedCsgAwithanti‐CsgAantibodyandfoundnosignalfromculturesofMG1655

PRO∆csgAompR234cells(“–ctrl”)oruninducedaTcReceiver/CsgAHiscells.Incontrast,

aTcReceiver/CsgAHiscellsinducedwith250ng/mlaTcgavenosignalat0h,butatlater

timepointsgavesignalcorrespondingto30‐40 gofCsgApercm2ofbiofilm(solidblue

line).Similarly,positivecontrolMG1655ompR234cells(“+ctrl”)gavenosignalat0h,

butatlatertimepointsgavesignalcorrespondingto40‐80 gofCsgApercm2ofbiofilm.

Atthe24htimepoint,wewerealsoabletousebiofilmvolumeasdeterminedby

confocalmicroscopy(Fig.1c)tocalculate63±5.8(s.e.m.)mgofCsgApercm3ofbiofilm

76


generatedbytheaTc‐inducedaTcReceiver/CsgAHiscells.Forcalculationofprotein

concentrationinsamples,areferencecurvewasconstructedusingserialdilutionsof

purifiedmatureCsgAwithouttheSecsignalsequencethatiscleavedoffduring

secretion.MatureCsgAwascheckedforpuritybyrunningonaSDS‐PAGEgelwith

Coomassiestaining.ThepurifiedCsgAmonomermigratesatahigherapparentMWon

thegel,at~17.5kDa,eventhoughtheactualMWis14.2kDa;thisisconsistentwith

resultsreportedintheliterature1.

77

SupplementaryFigureA‐23|Plasmidmaps

78


79


80


81


82


83


84


85


86

SupplementaryMethods

Plasmidconstruction.Theplasmidsusedinthisstudywereconstructedwithstandard

molecularcloningtechniques2usingNewEnglandBiolabs(NEB)restriction

endonucleases,T4DNALigase,andPhusionPCRkits.ABio‐RadS1000ThermalCycler

withDual48/48FastReactionModules(Bio‐Rad)wasusedtoperformPCRs,ligations,

andrestrictiondigests.GelextractionswerecarriedoutwithQIAquickGelExtraction

Kits(Qiagen).CustomoligonucleotideprimerswereobtainedfromIntegratedDNA

Technologies(Coralville,IA).

AllligationsforplasmidconstructionweretransformedintoE.colistrain

DH5αPROwithstandardprotocols2.IsolatedcolonieswereinoculatedintoLuria‐

Bertani(LB)‐Millermedium(Fisher),whichisLBmediumwith10g/LNaCl2,using

antibioticscorrespondingtoantibiotic‐selectionmarkerswithintheplasmids

(SupplementaryTables1,2)atthefollowingconcentrations:carbenicillinat50μg/ml,

chloramphenicolat25μg/ml,kanamycinat30μg/ml,andspectinomycinat100μg/ml.

DNAwasextractedwithQiagenQIAprepSpinMiniprepKits.Plasmidconstruct

sequenceswereconfirmedbyrestrictiondigestandsequencingwasperformedby

Genewiz(Cambridge,MA).

Thepartsthatconstitutetheplasmidsusedinthisworkaredescribedin

SupplementaryTable1,andplasmidsaredescribedinSupplementaryTable2.To

createconstructsfortheexpressionofoutputgenesundertightregulationbyanaTc‐

inducibleriboregulator,pZE‐AmpR‐rr12‐pL(tetO)‐gfpwasusedasastartingpoint3.The

AmpRcassetteofpZE‐AmpR‐rr12‐pL(tetO)‐gfpwasreplacedbytheCmRcassettefrom

87

pZS‐CmR‐pL(lacO)‐gfpbyusingSacI‐HF/AatIIdigestionandT4ligationtocreatepZE‐

CmR‐rr12‐pL(tetO)‐gfp.Then,theColE1originofpZE‐CmR‐rr12‐pL(tetO)‐gfpwas

replacedbythep15AoriginfrompZA‐AmpR‐pL(tetO)‐gfpbyusingSacI‐HF/AvrII

excisionandT4ligationtocreatepZA‐CmR‐rr12‐pL(tetO)‐gfp.Thegfpgenewasexcised

usingKpnI/MluIdigesttocreatethevectorpZA‐CmR‐rr12‐pL(tetO)‐.ThegenescsgA,

csgAHis,mCherry,andcsgAFLAGwithKpnIandMluIstickyendsweregeneratedbyPCR

andKpnI/MluIdigest;thesefragmentswereligatedwithpZA‐CmR‐rr12‐pL(tetO)‐to

createpZA‐CmR‐rr12‐pL(tetO)‐csgA,pZA‐CmR‐rr12‐pL(tetO)‐csgAHis,pZA‐CmR‐rr12‐

pL(tetO)‐mCherry,andpZA‐CmR‐rr12‐pL(tetO)‐csgAFLAGplasmids,respectively.

Tocreateconstructsfortheexpressionofoutputgenesundertightregulationby

anAHL‐inducibleriboregulator,pZE‐KanR‐rr12y‐pLuxR‐gfpwasusedasastarting

point3.TheKanRcassetteofpZE‐KanR‐rr12y‐pLuxR‐gfpwasreplacedbytheCmR

cassettefrompZS‐CmR‐pL(lacO)‐gfpbyusingSacI‐HF/AatIIdigestionandT4ligationto

createpZE‐CmR‐rr12y‐pLuxR‐gfp.Then,theColE1originofpZE‐CmR‐rr12y‐pLuxR‐gfp

wasreplacedbythep15AoriginfrompZA‐AmpR‐pL(tetO)‐gfpbyusingSacI‐HF/AvrII

excisionandT4ligationtocreatepZA‐CmR‐rr12y‐pLuxR‐gfp.Thegfpgenewasexcised

usingKpnI/MluIdigesttocreatethevectorpZA‐CmR‐rr12y‐pLuxR‐.Acodon‐optimized

geneencodingaCsgAvariantconsistingofeighttandemrepeatsofCsgAwithahistidine

tagattheC‐terminus(8XcsgAHis),andflankedbyKpnIandMluIsites,wasdesignedand

customsynthesizedbyGenScript(Piscataway,NJ).Thegene8XcsgAHiswithstickyends

wasgeneratedbyKpnI/MluIdigestandligatedwithpZA‐CmR‐rr12y‐pLuxR‐tocreate

pZA‐CmR‐rr12y‐pLuxR‐8XcsgAHis.ThegenescsgA,csgAHis,andcsgASpyTag,alsowithKpnI

andMluIstickyends,wereligatedwiththevectortocreatepZA‐CmR‐rr12y‐pLuxR‐

88

csgA,pZA‐CmR‐rr12y‐pLuxR‐csgAHis,andpZA‐CmR‐rr12y‐pLuxR‐csgASpyTag,

respectively.

AplasmidwithanAmpRresistancemarkerwasalsocreatedforuseinco‐culture

experiments(Fig.3).Thisplasmidhadalow‐copyoriginandanoutputgene(lacZalpha)

undertightrepressionbyariboregulatortoensureminimaleffectsonthehostcell

apartfromconferringampicillinresistance.TheplasmidpZE‐KanR‐rr10‐pL(lacO)‐

mCherrywasusedasastartingpoint3.TheKanRcassetteofpZE‐KanR‐rr10‐pL(lacO)‐

mCherrywasreplacedbytheAmpRcassettefrompZA‐AmpR‐pL(tetO)‐gfpbyusing

SacI‐HF/AatIIdigestionandT4ligationtocreatepZE‐AmpR‐rr10‐pL(lacO)‐mCherry.

Then,theColE1originofpZE‐AmpR‐rr10‐pL(lacO)‐mCherrywasreplacedbythe

pSC101originfrompZS‐CmR‐pL(lacO)‐gfpbyusingSacI‐HF/AvrIIexcisionandT4

ligationtocreatepZS‐AmpR‐rr10‐pL(lacO)‐mCherry.ThemCherrygeneofpZSAmpR‐

rr10‐pL(lacO)‐mCherrywasreplacedbythelacZalphagenefrompZE‐AmpR‐pLuxR‐

lacZalphausingKpnI/MluIexcisionandT4ligationtocreatepZS‐AmpR‐rr10‐pL(lacO)‐

lacZalpha.

PlasmidsforexpressinggenesfromthepL(lacO)promoterwereconstructedby

usingpZS‐CmR‐pL(lacO)‐gfp4asastartingpoint.Thegfpgenewasexcisedbydigestion

withKpnI/MluItocreatevectorpZS‐CmR‐pL(lacO)‐.Thisvectorwasligated,usingT4

ligase,withdigestedPCRfragmentsthathadKpnIandMluIstickyends.These

fragmentsencodedcsgA,csgAHisafterC‐terminus,orcsgAZnSpeptide.ThiscreatedpZS‐CmR‐

pL(lacO)‐csgA,pZS‐CmR‐pL(lacO)‐csgAHisafterC‐terminus,andpZS‐CmR‐pL(lacO)‐csgAZnS

peptideplasmids.

ThepDEST14‐T7‐CCSpyCatcherplasmidfortheexpressionofCCSpyCatcherwas

createdfrompDEST14‐T7‐SpyCatcher5usingtheQuikChangeLightningKit(Agilent)to

89

addcodonsencodingtwocysteinesimmediatelyafterthestartcodon.Thethiolgroups

oncysteineswereusedtoconjugateSpyCatchertoQDsasdescribedbelow.

Strainconstruction.TocreatetheE.coliMG1655PRO∆csgAompR234strainusedin

thisstudy,wesequentiallygeneratedE.coliMG1655PRO∆csgA::aph,E.coliMG1655

PRO∆csgA,andE.coliMG1655PRO∆csgAompR234.ThePROcassette(Placiq/lacI,

PN25/tetR,SpecR)hasaspectinomycinresistancemarkerandexpresseslacIandtetRat

highconstitutivelevels4.

E.coliMG1655PRO∆csgA::aphwasgeneratedviaP1transductionofthe

∆csgA::aphmutationfromdonorstrainJW1025‐1oftheKeiocollection6intorecipient

E.coliMG1655PRO3,aspreviouslydescribed2.ToprepareP1lysate,100μlovernight

cultureofdonorstrainwasincubatedwith100μlP1phagein10mlLB+7mMCaCl2+

12mMMgSO4for20minwithoutshakingat37°C,thenfor2hwithshakingat37°C.To

producemoreP1lysate,300μlmoreofdonorstrainwasadded,thecultureincubated

for20minwithoutshakingat37°C,thenfor2hwithshakingat37°C.Theculturewas

transferredtoa50mlFalcontube,2mlofchloroformwasadded,andthemixturewas

vortexedandcentrifugedfor15minat3000rpm.Thesupernatant,containingtheP1

lysate,wastransferredtoEppendorftubesandcentrifugedfor5minat14000rpmto

pelletremainingdebris.Fortransduction,theP1lysatewasdiluted60‐foldinto1mlLB

+7mMCaCl2+12mMMgSO4inanEppendorftube,100μlovernightcultureofrecipient

strainwasadded,andthetubewasincubatedfor1hwithshakingat37°C.Thisculture

wasthenplatedontoLBkanamycinagarwith25mMsodiumcitratetoselectfor

recipientssuccessfullytransducedwiththe∆csgA::aphmutation.Transformantswere

restreakedontoLBkanamycinagarforre‐isolation.

90

E.coliMG1655PRO∆csgAwasgeneratedbyremovingthekanamycinresistance

cassetteaspreviouslydescribed7(inordertofreethisantibioticselectionmarkerfor

subsequentusage).Briefly,MG1655PRO∆csgA::aphwastransformedwithpCP208

carryingthegeneforFLPrecombinaseandselectedonLBampicillinagarat30°C.

Transformantswererestreakedontonon‐selectiveLBandgrownovernightat42°Cto

curethetemperature‐sensitivepCP20.IsolateswerepatchedontoLBagarcontaining

eitherkanamycinorampicillintoconfirmthelossoftheFRT‐flankedkanamycin

cassetteandpCP20,respectively.

Finally,E.coliMG1655PRO∆csgAompR234wasgeneratedviaP1transduction

oftheompR234mutation(linkedtoakanamycinresistancemarker)fromdonorE.coli

MG1655ompR2349intorecipientE.coliMG1655PRO∆csgAwiththesameprotocolas

above.ThefinalstrainwasverifiedviaPCRampliconsizeusingcheckprimersforeach

locus.TheompR234mutationwasverifiedviasequencingofbothstrandsofthe

amplicon.WealsousedsequencingtoverifythatthecsgBAClocusinMG1655PRO

∆csgAompR234perfectlymatchedthepredictedsequenceforthecsgAknockout,with

anintactcsgBgene,asconstructedintheKeiocollection6(SupplementaryFig.20).

Finally,cellstrainsusedinpatterningexperimentsinthisstudywerecreatedby

transformingplasmidsconstructedaboveintoMG1655PRO∆csgAompR234,andare

describedinSupplementaryTable3.

Cultureconditions.Seedcultureswereinoculatedfromfrozenglycerolstocksand

growninLB‐Millermediumusingantibioticscorrespondingtotheplasmidsinthecells

(SupplementaryTable3)atthefollowingconcentrations:carbenicillinat50μg/ml,

chloramphenicolat25μg/ml,kanamycinat30μg/ml,andspectinomycinat100μg/ml.

Seedculturesweregrownfor12hat37°Cin14‐mlculturetubes(Falcon),withshaking

91

at300rpm.ExperimentalculturesweregrowninM63minimalmedium(Amresco)

supplementedwith1mMMgSO4andwith0.2%w/vglucoseor0.2%w/vglycerol

(hereafterreferredtoasglucose‐supplementedM63orglycerol‐supplementedM63,

respectively);allliquidexperimentalculturesweregrownin24‐wellplatewellsat30°C

withnoshaking10.Forinducingconditions,inducersusedwereanhydrotetracycline

(Sigma)atconcentrationsof1‐250ng/mlandN‐(β‐ketocaproyl)‐L‐homoserinelactone

(Sigma)atconcentrationsof1‐1000nM.Allexperimentsreportingstandard‐error‐of‐

the‐mean(s.e.m.)errorbarswereperformedintriplicate.

Forexperimentstoshowinduciblebiofilmformationandinduciblecurli

production(Fig.1;SupplementaryFigs.1,3,8),thefollowingstrainsandgrowth

conditionswereused:aTcReceiver/CsgAHisataninitialseedingdensityof5X107cells/ml

wasgrownonThermanoxorpolystyreneorembeddedin0.7%M63agar,for24hinthe

caseofliquidmediaand48hinthecaseofagar,withglucoseorglycerol‐supplemented

M63,withnoaTcorwith250ng/mlaTc.PositivecontrolstrainMG1655ompR234and

negativecontrolstrainMG1655PRO csgAompR234weresimilarlyculturedinM63

media,withoutinducer.

Forexperimentstoshowinduciblebiofilmformationbyconfocalmicroscopy

(Fig.1;SupplementaryFigs.2,14),thesameconditionswereusedasabove,withthe

cellstrainsusedpossessinganadditionalconstitutivemCherry‐expressingplasmid,and

usingThermanoxcoverslips.TheinduciblestrainusedwasaTcReceiver/CsgAHiswiththe

additionofpZE‐AmpR‐proB‐mCherry,thepositivecontrolstrainusedwasMG1655

ompR234/pZE‐AmpR‐proB‐mCherry,andthenegativecontrolstrainusedwasMG1655

PRO csgAompR234/pZE‐AmpR‐proB‐mCherry.Culturesweregrownfor4‐48h.

92

Biofilmsinflowcells(Fig.1;SupplementaryFig.2).PDMS(Polydimethylsiloxane;

Sylgard184;DowCorning,MI,USA)flowdevicesweremoldedfromapolystyrene

masteryieldinganegativeimprintof4straightchannels(2mmdeep/2mm

wide/350mmlong)andthenbondedwithsiliconeadhesivetoThermanoxcoverslips

(Nunc).Foreachstrainandexperimentalconditiontested,abacterialsuspensionof

5X107cells/mlinglucoseorglycerolsupplementedM63wasintroducedintoflowcells

undercontinuousflowdrivenbysyringepumps(PHDUltra,HarvardApparatus,MA,

USA)ataflowrateof0.5µl/min.DeviceswereplacedonaninvertedZeiss510Meta

confocallaser‐scanningmicroscopeforthedurationoftheexperiments.Fluorescence

imageswererecordedacrossthechannelsat4,10,24,and48hoursusinga10x/0.45

n.a.objective.ThetotalbiomassforeachexperimentwascalculatedinImageJ11for2

dimensionalsurfacecoverage(xy)andbyCOMSTATforMATLAB12forbiofilm

formation(xyzimageseries).Foreachdatapoint,3z‐stackspersamplewereanalysed

across3samples.

Staticculturebiofilms(Fig.1;SupplementaryFig.2).Biofilmformationinstatic

culturewasquantifiedonThermanoxcoverslipsat4and24hours.Eachsamplewas

rinsedin1XPBStoremoveunattachedcells,placedonaNo.1coverslipandimaged

withaninvertedZeiss510Metaconfocallaser‐scanningmicroscopeusinga100x/1.4

n.a.oilimmersionobjective.Fluorescenceimageswererecordedatmultiplelocations

acrossthesubstratetoquantifytheaveragebiomasscoverageforeachsubstrate.

Biomasswasquantifiedasdescribedforflowcellexperiments.Foreachdatapoint,3z‐

stackspersamplewereanalysedacross3samples.

Transmissionelectronmicroscopy.Fortransmissionelectronmicroscopy(TEM),a

20µldropletofsamplewasplacedonparafilm(Pechiney),anda200‐mesh

93

formvar/carboncoatednickelTEMgrid(ElectronMicroscopySciences)wasplaced

withcoatedsideface‐downonthedropletfor30‐60s.Inallprotocolsrequiring

incubationforgreaterthan10min,sampleswerecoveredwithpetridishlidsto

minimizeevaporation.ThegridwasthenrinsedwithddH2Obyplacingthegridface‐

downina30µldropletofddH2Oandwickingoffonfilterpaper(Whatman),andplaced

face‐downfor15‐30sonadropletof2%uranylacetate(UA)(ElectronMicroscopy

Sciences)filteredthrough0.22µmsyringefilter(Whatman).Excessuranylacetatewas

wickedoffandthegridwasallowedtoairdry.TEMimageswereobtainedonaFEI

TecnaiSpirittransmissionelectronmicroscopeoperatedat80kVacceleratingvoltage.

High‐resolutiontransmissionelectronmicroscopy(HRTEM)andenergy‐dispersiveX‐

rayspectroscopy(EDS)wereperformedonaJEOL2010Felectronmicroscope

operatingat200kV.

Scanningelectronmicroscopy.Forscanningelectronmicroscopy(SEM),biofilm

samplesoncoverslipswerecoatedwithcarbonsputteredto~10nmwithaDeskII

SputterCoater(DentonVacuum).ThesampleswerethenimagedwithaJEOLJSM‐

6010LAscanningelectronmicroscopeoperatedat10kVacceleratingvoltage.Images

wereobtainedinsecondaryelectronimaging(SEI)mode,andelementalmappingwas

performedwithenergy‐dispersiveX‐rayspectroscopy(EDS).

Fluorescencemicroscopy.Fluorescence‐lifetimeimagingmicroscopy(FLIM)was

performedwithaZeiss710NLOmultiphotonmicroscopewith20Xobjectiveand

connectedtoatime‐correlatedsingle‐photoncountingsystem(Becker&Hickl).The

excitationsourcewasa2‐photonlaser(CoherentChameleonVisionII)tunedto800nm,

andemissionwasdetectedthrougha590‐650nmbandpassfilter.FLIMdecayswerefit

usingSPCImagesoftware(BeckerandHickl,Germany).

94

LambdascananalysisoffluorescentZnSnanocrystalswasperformedwitha

ZeissLSM710NLOLaserScanningConfocalwith10Xobjectiveand405nmexcitation

laser.

Anti‐CsgAimmuno‐labellingassay(Fig.1,SupplementaryFig.1).Weoptimized

antibodyconcentrationsandbasedourprotocolonthatusedinCollinsonetal.13.TEM

grids(ElectronMicroscopySciences)wereplacedwithcoatedsideface‐downon20µl

dropletsofsamplesonparafilmfor2min.ThesideoftheTEMgridswithsamplewas

rinsedwitha30µldropletofddH2O,thenplacedona50µldropletofBlockingBuffer

(10mMTris(pH8.0)‐0.15MNaCl‐1%skimmilk)for30min,followedbytransfertoa

50µldropletofBindingBuffer(10mMTris(pH8.0)‐0.15MNaCl‐0.1%skimmilk)with

1:1000dilutionofrabbitanti‐CsgAantibody(M.Chapman,UniversityofMichigan14,15),

whereitwasincubatedfor1hatroomtemperature(RT).Thegridwasthenrinsedfour

timesin50µldropletsofWashBuffer(10mMTris(pH8.0)‐0.15MNaCl),and

subsequentlytransferredtoa50µldropletofBindingBufferwith1:10dilutionofgoat

anti‐rabbitantibodyconjugatedto10nmgoldparticles(Sigma),whereitwasincubated

for1hatRT.Thegridwasthenrinsedfourtimesin50µldropletsofWashBuffer,

followedbyfourrinsesin30µldropletsofddH2O.Thethoroughlywashedgridwas

placedface‐downonadropletoffiltered2%uranylacetatefor15‐30stonegativestain

thesample.Excessuranylacetatewaswickedoffwithfilterpaperandthegridwas

allowedtoairdry.AllsamplepreparationstepsweredoneatRT.

NiNTA‐AuNPlabellingassay.Fornickelnitrilotriaceticacidgoldnanoparticle(NiNTA‐

AuNP)labellingofhistidinetagsdisplayedonCsgA,200‐meshformvar/carbon‐coated

nickelTEMgrids(ElectronMicroscopySciences)wereplacedwithcoatedsideface‐

downon20µldropletsofsamplesonparafilmfor2min.ThesideoftheTEMgridwith

95

samplewasrinsedwitha30µldropletofddH2O,thenwithselectivebindingbuffer

(1XPBSwith0.487MNaCl,80mMimidazole,and0.2v/v%Tween20),andplacedface‐

downina60µldropletofselectivebindingbufferwith10nM5nmNiNTA‐AuNP

particles(Nanoprobes).TheTEMgridanddropletonparafilmwascoveredwithapetri

dishtominimizeevaporationandallowedtoincubatefor90min.Thegridwasthen

washed5timeswithselectivebindingbufferwithoutNiNTA‐AuNPparticles,thentwice

with1XPBSandtwicewithddH2O.Thethoroughlywashedgridwasplacedface‐down

onadropletoffiltered2%uranylacetatefor15‐30stonegativestainthesample.

Excessuranylacetatewaswickedoffwithfilterpaperandgridallowedtoairdry.All

samplepreparationstepsweredoneatRT.ImageswereobtainedonaFEITecnaiSpirit

transmissionelectronmicroscopeoperatedat80kVacceleratingvoltage.

AuNPbindingassay.ThisassaywasperformedidenticallytotheNiNTA‐AuNP

labellingassaywithtannic‐acid‐capped5nmAuNP(TedPella)inplaceofNiNTA‐AuNP.

Verifyingthespecificityofthehistidine‐tag‐NiNTAinteraction(Supplementary

Fig.9).StrainaTcReceiver/CsgAHiswasusedtoproduceCsgAHisfibrilsandstrain

aTcReceiver/CsgAwasusedtoproduceCsgAfibrils;cellswereseededat5X107cells/ml

andculturedinglucose‐supplementedM63mediawith50ng/mlaTcfor24hat30°C.

CsgAHisfibrilsandCsgAfibrilswerelabelledwiththeNiNTA‐AuNPlabellingassay;

additionallyCsgAHisfibrilswerelabelledwiththeAuNPparticlebindingassay.

Strainverificationexperiments(SupplementaryFigs.5,8).Forstrainverification

experiments,allcultureshadaninitialseedingconcentrationof5X107cells/mland

weregrowninM63mediumsupplementedwith0.2%w/vglucoseasacarbonsource.

ForexperimentstoverifythatthefibrilsseenonTEMwereself‐assembledfromCsgA,

MG1655ompR234wasgrownfor24h,aTcReceiver/CsgAwasgrownfor14hwith

96

62.5ng/mlaTc,andaTcReceiver/CsgAHiswasgrownfor14hwith62.5ng/mlaTc.Biofilms

resultingfromaTcReceiver/CsgAandaTcReceiver/CsgAHiscultureswereresuspendedin

1XPBSandtheNiNTA‐AuNPparticlelabellingassay(above)wasperformedto

characterizecurlifibrils.Forexperimentstoverifythatinsertionofhistidinetagsinto

CsgAstillallowscurlifibrilproduction(SupplementaryFig.5),aTcReceiver/CsgAwas

grownfor14hwith250ng/mlaTc,aTcReceiver/CsgAHiswasgrownfor14hwith250ng/ml

aTc,AHLReceiver/CsgAwasgrownfor14hwith1000nMAHL,andAHLReceiver/CsgAHiswas

grownfor14hwith1000nMAHL.Theresultingbiofilmswereresuspendedin1XPBS

andTEMimagingwasperformed.

ExperimentswereperformedtoverifythattheMG1655PRO∆csgAompR234

hoststraindoesnotproducecurlifibrils,thatAHLReceiver/CsgAHisonlyproducescurli

fibrilswheninducedbyAHL,andthataTcReceiver/CsgAHisonlyproducescurlifibrilswhen

inducedbyaTc(SupplementaryFig.8).MG1655PRO∆csgAompR234andMG1655

ompR234weregrownfor48h.AHLReceiver/CsgAHiswasgrownfor48hwithnoinducer,

1000nMAHL,or250ng/mlaTc.Similarly,aTcReceiver/CsgAHiswasgrownfor48hwithno

inducer,1000nMAHL,or250ng/mlaTc.Theresultingbiofilmswereresuspendedin

1XPBSandTEMimagingwasperformed.ImageJ(NIH)wasusedtothresholdimages

andcalculateareacoveredbycurlifibrilsandareacoveredbycells.Theratioofareas

wasusedtoquantifycurlifibrilproduction.

CrystalViolet(CV)biofilmassays(SupplementaryFig.3).Experimentswere

performedtoverifythataTcReceiver/CsgAHisformedthickadherentbiofilmsonlywhen

inducedbyaTc,onbothThermanoxcoverslips(Nunc)andonpolystyrene.Thermanox

coverslipswereplacedatthebottomof24‐wellplatewellsinwhichaTcReceiver/CsgAHis,

ataninitialseedingdensityof5X107cells/ml,wasgrownfor24hinglucose‐

97

supplementedM63withnoaTcorwith250ng/mlaTc,in24‐wellplatewellswithor

withoutThermanoxcoverslips.PositivecontrolstrainMG1655ompR234andnegative

controlstrainMG1655PRO csgAompR234weresimilarlyculturedfor24hinglucose‐

supplementedM63.

Crystalvioletstainingandquantificationwasperformedfollowingastandard

protocolgiveninO’Tooleet.al.16Thermanoxcoverslipswithbiofilmwerewashedin

ddH2Otoremoveunattachedcells,placedinaclean24‐wellplatewellwith400µlof

0.1%aqueouscrystalviolet(Sigma),andincubatedatRTfor10‐15min,afterwhichthe

coverslipswerewashedbyimmersing3‐4timesinatubofddH2Ountilnomorevisible

dyewaswashedoff.TheThermanoxcoverslipswereallowedtoairdry,placedback

intoclean24‐wellplatewells,andphotographstakenwithaChemiDocMPimaging

system(BioRad).ForquantificationofCVstaining,theThermanoxcoverslipswere

placedin24‐wellplatewellsand400µlof30%aceticacidinwaterwasadded.The

coverslipswereincubatedfor10‐15minatRTtosolubilizeCV.Subsequently,125µlof

solubilizedCVwastransferredtoa96‐wellplatewellandabsorbanceat550nmwas

measured,with30%aceticacidinwaterasablank.

Forbiofilmsgrownonpolystyrenesurfaceof24‐wellplatewells,washsteps

wereperformedbyrepeatedlyimmersingentireplatesinatubofddH2O.

CongoRedassayforquantificationofcurlifibrilproduction(SupplementaryFig.

4).Cellsweregrowntosaturationat30°CinliquidYESCAmedia(10g/Lcasamino

acids,1g/Lyeastextract)and15µlwasdropcastonYESCAagar(10g/Lcasaminoacids,

1g/Lyeastextract,and20g/Lagar)intriplicateandgrownfor72hat30°C17.Colonies

werescrapedandresuspendedin700µl1XPBS;300µlofcellsuspensionwasusedfor

OD600cellnumbernormalization,andtheremaining400µlcombinedwith5XCongo

98

Red(CR)forafinalconcentrationof20µg/mlCR,andincubatedfor5minatRT.The

cellsandcurliwithboundCRwerespundownat15,000xgfor5min,and300µl

supernatantwasremovedforCRquantification.ConcentrationofCRinsupernatantwas

quantifiedbyabsorbanceat480nm.TheamountofCRboundbycellsandcurliwere

quantifiedbysubtractingtheAb480nmofsupernatantfromAb480nmof20µg/mlCR18.

ForgrowthofBW25113 csgA,kanamycinat30μg/mlwasaddedtoYESCA.For

growthofBW25113 csgA/pZS3‐pL(lacO)‐csgAandBW25113 csgA/pZS3‐pL(lacO)‐

csgAHisafterC‐terminus,chloramphenicolat25μg/mlandkanamycinat30μg/mlwereadded

toYESCA.

EvaluatingpL(lacO)forregulationofcurliproduction(SupplementaryFig.19).

Cellsgrowntosaturationat30°CinliquidYESCAmediaweredropcastontoYESCA

agarandgrownfor48hat30°C17.ForgrowthofBW25113 csgA,kanamycinat

30μg/mlwasaddedtoYESCA.ForgrowthofBW25113 csgA/pZS3‐pL(lacO)‐csgA+

pZS4Int‐lacI/tetR,chloramphenicolat25μg/ml,spectinomycinat100μg/ml,and

kanamycinat30μg/mlwereaddedtoYESCA.Forisopropylβ‐D‐1‐

thiogalactopyranoside(IPTG)induction,1mMIPTGwasaddedtoYESCAagar.

Patterningexperiments(Figs.2‐4;SupplementaryFigs.6,7,10‐12).Inallcases,

culturesweregrownat30°Cwithnoshaking.Afterpatterning,theresultingcell

populationswereresuspendedin1XPBSandtheNiNTA‐AuNPlabellingassayandTEM

imagingwereperformedtocharacterizetheresultingamyloidfibrils.ImageJ(NIH)was

usedtomeasurethelengthofunlabelledandNiNTA‐AuNPlabelledfibrilsegments.

Fortunablepatterningbasedonchanginginductiontime(Fig.2a,b),

AHLReceiver/CsgAataninitialseedingdensityof5X106cells/mlandaTcReceiver/CsgAHisat

99

aninitialseedingdensityof5X105cells/mlwereco‐culturedin24‐wellplatewellswith

a12mmglasscoverslip(TedPella)placedatthebottom.Cellswereculturedinglycerol‐

supplementedM63media.Thecellswerefirstco‐culturedinthepresenceof50nMN‐

(β‐ketocaproyl)‐L‐homoserinelactone(AHL)inducerfor18‐48h.Themediawasthen

removedandtheresultingcellswereincubatedwithno‐inducermediafor6h.Theno‐

inducermediawasthenremovedandreplacedwithmediawith50ng/mlaTcinducer.

Thecellswereresuspendedandtheculturesincubatedfor16h.Theresultingcellswere

resuspendedandtheNiNTA‐AuNPlabellingassaywasperformedtocharacterizecurli

fibrils.ForthecontrolexperimenttoproducefibrilswhenCsgAandCsgAHiswere

secretedslowlyandsimultaneouslywithouttemporalseparation,AHLReceiver/CsgAand

aTcReceiver/CsgAHiswereco‐culturedfor24hinglycerol‐supplementedM63mediawith

inductionby50nMAHLand50ng/mlaTcsimultaneously.

Fortunablepatterningbasedonvaryinginducerconcentration(Fig.2c,d),

AHLReceiver/CsgAandaTcReceiver/CsgAHis,eachataninitialseedingdensityof5X107

cells/ml,wereco‐culturedinglucose‐supplementedM63mediawithAHLinducerat0‐

1000nMandaTcinducerat0‐250ng/ml,for18h.

Forproductionofadynamicmaterialwhosecompositionchangeswithtime(Fig.

3),acellularcommunicationsystemconsistingofAHLSender+aTcReceiver/CsgAand

AHLReceiver/CsgAHiswasused.Thetwocellstrainswereco‐cultured,with

AHLSender+aTcReceiver/CsgAataninitialseedingdensityof5X106cells/mland

AHLReceiver/CsgAHisataninitialseedingdensityof5X107,5X106,or5X105cells/ml.The

cellswereco‐culturedinglucose‐supplementedM63mediawith50ng/mlaTcfor4‐

36h.TheresultingcellswereresuspendedandtheNiNTA‐AuNPlabellingassaywas

performedtocharacterizecurlifibrils.Forcontrols,AHLSender+aTcReceiver/CsgAand

100

AHLReceiver/CsgAHisweregrownseparately.Ineachcase,thecellsweregrownfor16h

with50ng/mlaTcinduction,withaninitialseedingdensityof5X106cells/ml.

Forpatterningattwodifferentlengthscalesbycombininggeneticregulationof

subunitexpressionwithspatialinducergradients(Fig.4a,b),inducer‐responsivecells

weregrowninsolidphaseonagarwithopposingAHLandaTcinducergradients.

Inducergradientagarplateswerepreparedbyatwo‐stepprocess19.A100mmX100mm

squarepetridish(TedPella)waselevatedatoneendby1cm.Itwasfirstfilledby20ml

of1.5%agarglucose‐supplementedM63with50nMAHLandwasallowedtoharden

intoanagarwedge.Theplatewasthenlaidflatandfilledwith20mlof1.5%agar

glucose‐supplementedM63with50ng/mlaTc.Theplateswereleftfor12htoallow

diffusionofinducerstosetupaconcentrationgradient.Thesurfaceoftheagarwas

thenoverlaidwith5mlof0.7%agarglucose‐supplementedM63(top‐agar)embedded

withfourcellstrains–AHLReceiver/CsgA,aTcReceiver/CsgAHis,AHLReceiver/GFP,and

aTcReceiver/mCherry–eachatacelldensityof3.33X108cells/ml.Thetop‐agarwith

embeddedcellswasallowedtoharden,andtheplatewasincubatedat30°Cfor40h.

FluorescencewasimagedwithaChemiDocMPimagingsystem(BioRad)and

fluorescenceintensityvaluesextractedwithImageJ(NIH).Topagarwassampledand

resuspendedin1XPBS,andtheNiNTA‐AuNPlabellingassaywasperformedto

characterizecurlifibrils.Forthecontrolconditionwithnoinducergradients,thesame

procedurewascarriedoutbutthepetridishwasnotelevatedatoneendtocreateagar

wedges.

Forpatterningbyprotein‐levelengineering(Fig.4c),AHLReceiver/8XCsgAHiscells,

ataninitialseedingdensityof5X106cells/ml,wereculturedinglucose‐supplemented

M63with10nMAHLinducerfor28h.Theresultingcellswereresuspendedandthe

101

NiNTA‐AuNPlabellingassaywasperformedtocharacterizecurlifibrils.Forthecontrol

experiment,aTcReceiver/CsgAHisataninitialseedingdensityof1.5X106cells/mlwas

culturedinglucose‐supplementedM63with10ng/mlaTcinducerfor28h.

Forpatterningattwodifferentlengthscalesbycombininggeneticregulationof

subunitexpressionwithsubunit‐levelproteinengineering(Fig.4d),

AHLReceiver/8XCsgAHisataninitialseedingdensityof5X107cells/mland

aTcReceiver/CsgAHisataninitialseedingdensityof5X105cells/mlwerefirstco‐culturedin

glucose‐supplementedM63mediawith4nMAHLinducerfor36h.Themediawasthen

removedandtheresultingcellsincubatedwithno‐inducerglucose‐supplementedM63

mediafor6h.Theno‐inducermediawasthenremovedandreplacedwithglucose‐

supplementedM63mediawith1ng/mlaTcinducer.Thecellswereresuspendedand

thecultureswereincubatedfor18h.

Conductivebiofilmdemonstration(Fig.5,SupplementaryFigs.13‐17).Tocreate

interdigitatedelectrodes(IDEs)formeasurementofbiofilmconductance,custom

shadowingmasks(Tech‐Etch)withholesoftheappropriatedimensions

(SupplementaryFig.13)wereplacedoverThermanoxcoverslips.Goldwassputteredto

athicknessof~200nmwithaDeskIISputterCoater(DentonVacuum)andtheresulting

IDEsweretestedwithaKeithley4200picoammeterwithtwo‐pointprobetoconfirm

theabsenceofshortcircuits.

IDEswereplacedatthebottomof24‐wellplatewellsandcoveredwithglucose‐

supplementedM63mediawith250ng/mlanhydrotetracycline(aTc),aTcReceiver/CsgAHis

cellsat1X108cells/ml,and100nMNiNTA‐AuNPparticles.Thecultureswereincubated

at30°Cwithnoshakingfor24h.Forcontrolexperiments,eitheraTcwasexcluded,

AHLReceiver/CsgAHiscellswereusedinplaceofaTcReceiver/CsgAHiscells,orNiNTA‐AuNP

102

particleswereexcluded(Fig.5,SupplementaryFig.17).aTcReceiver/CsgAHiswiththe

additionofpZE‐AmpR‐proB‐mCherrywasusedtoshowbyconfocalmicroscopythat

biofilmformationisinducedbyaTcinpresenceof100nMNiNTA‐AuNP,withimages

takenat4hand24htimepoints(SupplementaryFig.14).Pleaseseeabovefordetailsof

confocalmicroscopymethods.

ToobtainsamplesofbiofilmsforTEM,asmallamountofbiofilmwasscraped

fromtheIDEsubstrateandresuspendedin1XPBS.IDEswerewashedbyrepeatedly

immersinginddH2O,laidonaflatsurface,andallowedtoairdryforthreedays.A

Keithley4200picoammeterwithtwo‐pointprobewasusedtocarryoutavoltage

sweep;avoltagedifferencewasappliedacrossthetwoprobesandthecurrent

measured.ResultingI‐Vcurveswerefittedtosimplelinearregressionlinesand

conductanceofthebiofilmwasobtainedfromtheslope(SupplementaryFig.15).SEM

imagingwasthenperformedtocharacterizeintactbiofilms;SEMimagingwas

performedafterconductancemeasurementsbecausethesampleswerecoatedwitha

conductivecarbonlayertofacilitateimaging(Fig.5b).

Goldnanowireandnanorodsynthesis(Fig.5c).StrainsaTcReceiver/CsgAHisand

AHLReceiver/CsgAwereseededeachataconcentrationof5X107cells/mlintoglucose‐

supplementedM63,andgrownfor24hat30°Cin24‐wellplates.Thecellswereinduced

with50ng/mlaTcalone,100nMAHLalone,orbothsimultaneously.Theresultingcells

withcurlifibrilswereresuspendedin1XPBS,depositedonTEMgrids,andlabelledwith

5nmNiNTA‐AuNPusingthespecificNiNTA‐AuNPbindingprotocolabove.Goldwas

specificallydepositedonNiNTA‐AuNPchainsusingtheGoldEnhance™EMkit

(Nanoprobes).Goldenhancementwasperformedfor2minfollowingkitinstructions,

resultingingoldnanowiresandnanorods.

103

ExpressionandpurificationofSpyCatcherprotein.E.coliBL21DE3pLysS/

pDEST14‐T7‐CCSpyCatcher(SupplementaryTable3)wasgrownfor12‐16hat37°Cin

LB‐Millerwith50µg/mlcarbenicillinand0.4mMIPTG5.TheHis‐taggedSpyCatcher

proteinwithtwoN‐terminalcyteineswaspurifiedwithNi‐NTASpinColumns(Qiagen)

followingthenativeproteinpurificationprotocoldescribedintheNi‐NTASpinKit

Handbook.Thebufferoftheelutionfractionwasexchangedfor1XPBSusing0.5ml

Amiconfiltercolumns(MWCO3kDa).

SynthesisofQDsandconjugationtoSpyCatcher.CdTe/CdSQDswitha

photoluminescenceemissionpeakat620nmweresynthesizedfollowingthemethod

describedinDengetal.20.TheQDswerepurifiedbyaddingisopropylalcohol,followed

bycentrifugationat15000rpmfor15minandsubsequentresuspensioninddH2O.The

concentrationoftheCdTe/CdSQDsandtheamountofadditionalshellprecursors

neededtoobtainspecificshellthicknesseswerecalculatedbyfollowingthemethod

describedinDengetal.:5µlCd(NO3)2(asCd2+source)stocksolution(25mM)and10µl

3‐mercaptopropionicacidstocksolution(25mM)werecombinedwiththeCdTe/CdS

QDsdispersedin100μlddH2O,vortexed,andgentlysonicatedina1.5mlEppendorf.

Next,20µlof2mg/mlofCCSpyCatcherproteinwasaddedandgentlyvortexed.ThepH

wastunedto12.2byaddingNaOH(1M).Thereactionmixturewasplacedonaheating

blockat90°Cfor30min,andthencooleddownbysubmergingthetubeinanicewater

bath.Thereactedsolutionwasloadedintoa0.5mlAmiconfilter(MWCO30kDa,EMD

Millipore),250μl1XPBSbufferwasaddedtothefilter,andthesamplewassubjectedto

centrifugationat7000rpmfor5min.Thewashing(eachwashwasperformedwith

350μlof1XPBSbuffer)andcentrifugation(7000rpmfor5min)stepswererepeated

threetimes.Thefinalproductwasre‐dispersedin100μl1XPBS.Thediameterofthe

104

CdTe/CdSquantumdotswas~4nm.Wemeasuredtheemissionspectrumof

CCSpyCatcherconjugatedCdTe/CdSQDswithaNanoLogSpectrometer(HORIBAJobin

Yvon)andfoundanemissionpeakat638nmwhenexcitedat450nm.Theparticleswere

alsovisiblyhighlyfluorescentwhenilluminatedbyaUVlampat365nm

(SupplementaryFig.21).

SpecificbindingofQD‐SpyCatchertocurlifibrils(Fig.6a).Curlifibrilsdepositedon

TEMgridswerefloatedon50µlofSpyCatcherBindingBuffer(1XPBS+350mMNaCl+

0.3v/v%Tween20)for5minandsubsequentlytransferredto50µlSpyCatcherBinding

Bufferwith1:333dilutionofQD‐SpyCatcher,followedbyincubationatRTfor45min.

Thegridwassubsequentlywashedfourtimeswith50µldropletsofSpyCatcherBinding

Buffer,thenwashedfourtimeswith30µldropletsofddH2O,followedbynegative

stainingwithUAifnosubsequentAuNPbindingwasperformed.Strain

AHLReceiver/CsgASpyTagwasusedtoproduceCsgASpyTagfibrilsandstrainAHLReceiver/CsgA

wasusedtoproduceCsgAfibrils;cellswereseededat5X107cells/mlandculturedin

glucose‐supplementedM63mediawith100nMAHLfor12hat30°C.

Specificbindingof40nm‐AuNP‐antibodyconjugatestocurlifibrils(Fig.6a).Curli

fibrilsdepositedonTEMgridswerefloatedona50µldropletofBlockingBuffer(10mM

Tris(pH8.0)‐0.15MNaCl‐1%skimmilk)for30min,followedbytransfertoa50µl

dropletofBindingBuffer(10mMTris(pH8.0)‐0.15MNaCl‐0.1%skimmilk)with1:250

dilutionofrabbitanti‐FLAGantibody(Sigma),whereitwasincubatedfor1hatRT.The

gridwasthenrinsedfourtimesin50µldropletsofWashBuffer(10mMTris(pH8.0)‐

0.15MNaCl),andsubsequentlytransferredtoa50µldropletofBindingBufferwith1:10

dilutionofgoatanti‐rabbitantibodiesconjugatedto40nmAuNPs(Abcam),whereit

wasincubatedfor3hoursatRT.Thegridwasthenrinsedfourtimesin50µldropletsof

105

WashBuffer,followedbyfourrinsesin30µldropletsofddH2Oandnegativestaining

withUA.StrainaTcReceiver/CsgAFLAGwasusedtoproduceCsgAFLAGfibrilsandstrain

aTcReceiver/CsgAwasusedtoproduceCsgAfibrils;cellswereseededat5X107cells/ml

andculturedinglucose‐supplementedM63mediawith50ng/mlaTcfor12hat30°C.

Co‐dockingofQDandAuNPtocurlifibrils(Fig.6b‐c).StrainsAHLReceiver/CsgASpyTag

andaTcReceiver/CsgAFLAGwereseededeachataconcentrationof5X107cells/mlinto

glucose‐supplementedM63media,andgrownwith12mmglasscoverslips(TedPella)

for12hat30°C.Thecellswereinducedwith100nMAHLalone,50ng/mlaTcalone,or

bothsimultaneously.Curliwasresuspendedin1XPBS,depositedonTEMgrids,andthe

specificQD‐SpyCatcherbindingprotocolwasperformed,followedbythespecificAuNP‐

antibodybindingprotocolandnegativestainingwithUA.ForFLIMcharacterization,the

QD‐bindingprotocolfollowedbytheAuNP‐bindingprotocolwasperformeddirectlyon

rinsedcoverslipswithattachedcellsandcurlifibrils,sansnegativestaining.

Zincsulphidenanocrystalsynthesis(Fig.6d‐e).Cellsweregrownfor12hinLB‐

Miller,washedin1XPBS,andresuspendedtoaconcentrationof1010cells/ml.5µlofcell

suspensionwasdropcastonYESCAagarandgrownfor30hat30°C.Forculturing

BW25113 csgA/pZS3‐pL(lacO)‐csgAandBW25113 csgA/pZS3‐pL(lacO)‐csgAZnS

peptidestrains,chloramphenicolat25μg/mlwasaddedtoYESCAagar.Thecolonywas

thenresuspendedin30µlddH2O.WeusedaZnSsynthesisprotocolbasedonthatgiven

inMaoet.al.21Specifically,10µlofcellandcurlisuspensionwasaddedto1mlof1µM

ZnCl2andincubatedatRTfor12h.Then,1µMNa2Swasadded,andsampleswere

incubatedat0°Cfor24hbypackinginiceandplacingina4°Croom.Sampleswere

subsequentlyallowedtoagefor12hatRTbeforebeingdepositedonTEMgridsfor

HRTEMandglasscoverslipsforfluorescencecharacterization.

106

ExpressionandpurificationofmatureCsgAHisnoSS(SupplementaryFig.22).For

theconcentrationreferencestandardforquantitatingCsgAproductioninbiofilmsby

dotblot,weusepurifiedCsgAHisnoSS,whichisthematureformofCsgAproteinwithout

thesecretionsignalsequencethatiscleavedduringsecretion.Wefollowedtheprotocol

describedinZhongetal.22.

Insummary,thepET11d‐T7vector(Novagen)wasdigestedatBamHIandNcoI

restrictionsites,andthecsgAHisnoSSgene(SupplementaryTable1)wasamplifiedby

PCRwithintroductionofcompatibleoverhangsforGibsonassembly.Tocreatethe

pET11d‐T7‐csgAHisnoSSplasmid(SupplementaryTable2),thedigestedvectorand

csgAHisnoSSPCRproductwereincubatedwithGibsonAssemblyMasterMix(NEB)at

50°Cfor1h,thentransformedintoE.coliDH5 .Thesequence‐verifiedpET11d‐T7‐

csgAHisnoSSplasmidwasthentransformedintoE.coliBL21T7ExpressIq(NEB)to

createtheBL21T7ExpressIq/pET11d‐T7‐csgAHisnoSSstrain(SupplementaryTable3)

forproteinexpression.

BL21T7ExpressIq/pET11d‐T7‐csgAHisnoSSwasgrowntoOD600~0.9inLB‐

Millercontaining50 g/mlcarbenicillinat37°C.Proteinexpressionwasinducedwith

0.5mMIPTGat37°Cfor1h.Cellswerecollectedbycentrifugationandthepellets

storedat−80°C.ThecellsweresubsequentlyresuspendedandlysedwithExtraction

Buffer(8Mguanidinehydrochloride(GdnHCl),300mMNaCl,50mMK2HPO4/KH2PO4,

pH7.2).Atotalof50mlofExtractionBufferwasusedforeachcellpelletfroma1.5litre

culture.Lysateswereincubatedat4°Cfor24h.Theinsolubleportionsofthelysates

wereremovedbycentrifugationat10,000xg.6mlofTALONcobaltresin(Clontech)

waswashedwithEquilibrationBuffer(300mMNaCl,50mMK2HPO4/KH2PO4,pH7.2)

andthesupernatantwasincubatedwithequilibratedTALONcobaltresinfor2hatRT.

107

ResinbindingtheHis‐taggedCsgAHisnoSSwasspundown,washedtwicewith20ml

EquilibrationBuffer,andloadedintoHisTALON™GravityColumns(Clontech).Theresin

wasfurtherwashedwith12mlofEquilibrationBufferpassedthroughcolumns.

Subsequently,WashBuffer(40mMimidazole,300mMNaCl,50mMK2HPO4/KH2PO4,pH

7.2)waspassedthroughcolumnsin5consecutivewashsteps,using6mlineachwash.

ThenElutionBuffer(300mMimidazole,300mMNaCl,50mMK2HPO4/KH2PO4,pH7.2)

waspassedthroughcolumnstoeluteCsgAHisnoSSin3consecutiveelutionsteps,using

6mlineachelution.Fractionsfromwashandelutionstepswerethenmixedwith

loadingsamplebuffer(Novex4XLDSSampleBuffer,NuPAGE)ina3:1ratioandheated

at90°Cfor10min.ThesampleswerethenloadedintoaNovex4‐12%Bis‐Trisgel

(NuPAGE)andranat165Vfor35mininMESBuffer(NuPAGE).Thegelwasthenstained

withCoomassieBlueG‐250(0.1%in7/50/43aceticacid/methanol/ddH2O)by

incubatingatRTfor30min,followedby3consecutivewashstepswithincubationin

25mldestainingsolution(10/40/50aceticacid/methanol/ddH2O)for30minatRT,

followedbyincubationin25mlddH2OovernightatRT.Thedestainedgelwasthen

imagedwithaGelDoc™XR+SystemwithImageLab™Software(Bio‐rad).

Dotblotquantitationofcurliproduction(SupplementaryFig.22).StrainsMG1655

ompR234,MG1655PRO∆csgAompR234,andaTcReceiver/CsgAHiswereculturedinM63

glucoseat30°Cin24‐wellplatewells,withoutshaking,for0,12,and24h.

aTcReceiver/CsgAHiscellsweregrowneitherinthepresenceorabsenceof250ng/mlaTc.

Ateachtimepoint,culturesupernatantwasremovedwithoutdisturbingbiomassatthe

bottomofwells,andbiomassatthebottomofwellswasresuspendedin1mlddH2O.

CsgAHiswasquantifiedbyastandarddotblotprotocol,followingreferencesinwhich

dotblotswereusedtoquantitatetheconcentrationsofCsgAandotheramyloid

108

proteins23‐25.Briefly,2 lofsampleswerespottedontoProtranBA83nitrocellulose

membranes(Whatman)withadotblotmanifold(Schleicher&SchuellMinifold‐IDot‐

BlotSystem).ThemembranewasblockedinTBST+5%skimmilkfor30minatRT,

followedbyincubationinTBST+0.1%skimmilkwitha1:10000dilutionofrabbitanti‐

CsgAantibody(M.Chapman,UniversityofMichigan14,15)for1hatRT.Themembrane

wasthenwashedthreetimesinTBST(incubatingatRTfor5min),followedby

incubationinTBST+0.1%skimmilkwitha1:5000dilutionofHRP‐conjugatedgoat

anti‐rabbitIgGantibody(Abcam)for30minatRT.Subsequently,themembranewas

washedthreetimesinTBST(incubatingatRTfor15min,then2X5min),followedby

onewashinTBS(incubatingatRTfor5min).Thewashedmembranewasincubated

withSuperSignalWestPico(Thermo)chemiluminescentsubstratefollowingkit

instructions,andimagedwithaChemiDocMPimagingsystem(BioRad),with

quantitationofspotluminescencesignalintensitybyQuantityOne(BioRad)software.

Proteinconcentrationswerecalculatedfromareferencecurveestablishedbyserial

dilutionsoffibrilsofpurifiedmatureCsgAHisnoSSprotein,whichlackstheCsgA

secretionsignalsequencecleavedoffduringsecretion26.Thepurifiedproteinwas

incubatedatRTfor~12htoallowforfibrillization.Weaccountedforthedifferencein

molecularweightsbetweensecretedCsgAHis(15kDa)andpurifiedCsgAHisnoSS

(14.2kDa),sincetheformerhasanadditionalHistag.TheamountofCsgAHisproteinper

biofilmsamplegrownin24‐wellplatewellswasdividedbythe1.9cm2surfaceareaof

thewellstoobtain gofCsgAHispercm2ofbiofilm.Forthe24htimepoint,forwhichthe

volumeperunitareaofbiofilmwasmeasuredbyconfocalmicroscopy,wealso

calculatedmgofCsgAHispercm3ofbiofilm.

109

SupplementaryTables

SupplementaryTableA‐1|Syntheticpartsusedinthiswork.

PartName PartType Sequence Sour

ce

pL(tetO) Promoter TCCCTATCAGTGATAGAGATTGACATCCCTATCA

GTGATAGAGATACTGAGCACATCAGCAGGACGCA

CTGACC

4

pLuxR

Designated

“pLuxI”in3

Promoter ACCTGTAGGATCGTACAGGTTTACGCAAGAAAAT

GGTTTGTTATAGTCGAATA

3

pL(lacO) Promoter AATTGTGAGCGGATAACAATTGACATTGTGAGCG

GATAACAAGATACTGAGCACA

4

P(lac) Promoter CCGGTTAGCGCTCTCATTAGGCACCCCAGGCTTGA

CACTTTATGCTTCCGGCTCGTATAATGACTGCATT

TATTGGTAC

27

cr_rr10 Riboregulato

rcis

repressor

sequence

TACCTATCTGCTCTTGAATTTGGGT 3

cr_rr12 Riboregulato TACCATTCACCTCTTGGATTTGGGT 3

110

rcis

repressor

sequence

cr_rr12y Riboregulato

rcis

repressor

sequence

TACCATTCACCTCTTGGATTTAGCT 3

taRNA_rr10 Riboregulato

rtrans

activator

sequence

ACCCAAATTCATGAGCAGATTGGTAGTGGTGGTT

AATGAAAATTAACTTACTACTACCTTTC

3

taRNA_rr12 Riboregulato

rtrans

activator

sequence

ACCCAAATCCAGGAGGTGATTGGTAGTGGTGGTT

AATGAAAATTAACTTACTACTACCATATATC

3

taRNA_rr12

y

Riboregulato

rtrans

activator

sequence

AGCTAAATCCAGGAGGTGATTGGTAGTGGTGGTT

AATGAAAATTAACTTACTACTACCATATATC

3

rrnBT1 Terminator GGCATCAAATAAAACGAAAGGCTCAGTCGAAAGA

CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA

ACGCTCTCCTGAGTAGGACAAATCCGCCGCCCTAG

3

111

A

rrnBT Terminator

(double

terminator

fromrrnB)

TGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGAC

CCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGC

CGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTA

GGGAACTGCCAGGCATCAAATAAAACGAAAGGCT

CAGTCGAAAGACTGGGCCTT

28

luxR Repressor ATGAAAAACATAAATGCCGACGACACATACAGAA

TAATTAATAAAATTAAAGCTTGTAGAAGCAATAA

TGATATTAATCAATGCTTATCTGATATGACTAAA

ATGGTACATTGTGAATATTATTTACTCGCGATCA

TTTATCCTCATTCTATGGTTAAATCTGATATTTC

AATTCTAGATAATTACCCTAAAAAATGGAGGCAA

TATTATGATGACGCTAATTTAATAAAATATGATC

CTATAGTAGATTATTCTAACTCCAATCATTCACC

AATTAATTGGAATATATTTGAAAACAATGCTGTA

AATAAAAAATCTCCAAATGTAATTAAAGAAGCGA

AAACATCAGGTCTTATCACTGGGTTTAGTTTCCC

TATTCATACGGCTAACAATGGCTTCGGAATGCTT

AGTTTTGCACATTCAGAAAAAGACAACTATATAG

ATAGTTTATTTTTACATGCGTGTATGAACATACC

ATTAATTGTTCCTTCTCTAGTTGATAATTATCGA

AAAATAAATATAGCAAATAATAAATCAAACAACG

ATTTAACCAAAAGAGAAAAAGAATGTTTAGCGTG

GGCATGCGAAGGAAAAAGCTCTTGGGATATTTCA

3

112

AAAATATTAGGCTGCAGTGAGCGTACTGTCACTT

TCCATTTAACCAATGCGCAAATGAAACTCAATAC

AACAAACCGCTGCCAAAGTATTTCTAAAGCAATT

TTAACAGGAGCAATTGATTGCCCATACTTTAAAA

ATTAA

luxI AHL

signalling

molecule

generator

ATGACTATAATGATAAAAAAATCGGATTTTTTGG

CAATTCCATCGGAGGAGTATAAAGGTATTCTAAG

TCTTCGTTATCAAGTGTTTAAGCAAAGACTTGAG

TGGGACTTAGTTGTAGAAAATAACCTTGAATCAG

ATGAGTATGATAACTCAAATGCAGAATATATTTA

TGCTTGTGATGATACTGAAAATGTAAGTGGATGC

TGGCGTTTATTACCTACAACAGGTGATTATATGC

TGAAAAGTGTTTTTCCTGAATTGCTTGGTCAACA

GAGTGCTCCCAAAGATCCTAATATAGTCGAATTA

AGTCGTTTTGCTGTAGGTAAAAATAGCTCAAAGA

TAAATAACTCTGCTAGTGAAATTACAATGAAACT

ATTTGAAGCTATATATAAACACGCTGTTAGTCAA

GGTATTACAGAATATGTAACAGTAACATCAACAG

CAATAGAGCGATTTTTAAAGCGTATTAAAGTTCC

TTGTCATCGTATTGGAGACAAAGAAATTCATGTA

TTAGGTGATACTAAATCGGTTGTATTGTCTATGC

CTATTAATGAACAGTTTAAAAAAGCAGTCTTAAA

TTAA

27

113

csgA Curliamyloid

material

subunit

ATGAAACTTTTAAAAGTAGCAGCAATTGCAGCAA

TCGTATTCTCCGGTAGCGCTCTGGCAGGTGTTGTT

CCTCAGTACGGCGGCGGCGGTAACCACGGTGGTG

GCGGTAATAATAGCGGCCCAAATTCTGAGCTGAA

CATTTACCAGTACGGTGGCGGTAACTCTGCACTTG

CTCTGCAAACTGATGCCCGTAACTCTGACTTGACT

ATTACCCAGCATGGCGGCGGTAATGGTGCAGATG

TTGGTCAGGGCTCAGATGACAGCTCAATCGATCT

GACCCAACGTGGCTTCGGTAACAGCGCTACTCTTG

ATCAGTGGAACGGCAAAAATTCTGAAATGACGGT

TAAACAGTTCGGTGGTGGCAACGGTGCTGCAGTT

GACCAGACTGCATCTAACTCCTCCGTCAACGTGAC

TCAGGTTGGCTTTGGTAACAACGCGACCGCTCATC

AGTACTAA

1

csgAHis Curliamyloid

material

subunit




GCGGTAATAATAGCGGCCCAAATCACCATCACCA

TCACCACCATTCTGAGCTGAACATTTACCAGTACG

GTGGCGGTAACTCTGCACTTGCTCTGCAAACTGAT

GCCCGTAACTCTGACTTGACTATTACCCAGCATGG

CGGCGGTAATGGTGCAGATGTTGGTCAGGGCTCA

GATGACAGCTCAATCGATCTGACCCAACGTGGCT

TCGGTAACAGCGCTACTCTTGATCAGTGGAACGG

This

work

114

CAAAAATTCTGAAATGACGGTTAAACAGTTCGGT

GGTGGCAACGGTGCTGCAGTTGACCAGACTGCAT

CTAACTCCTCCGTCAACGTGACTCAGGTTGGCTTT

GGTAACAACGCGACCGCTCATCAGTACCACCATCA

CCATCACCACCATTAA

8XcsgAHis Curliamyloid

material

subunit

ATGAAGCTGCTGAAGGTGGCTGCTATCGCTGCTA

TCGTGTTTTCCGGTTCGGCACTGGCTGGTGTCGTC

CCGCAATACGGTGGCGGTGGCAACCATGGCGGTG

GCGGTAACAATAGTGGTCCGAACTCCGAACTGAA

TATTTATCAGTACGGCGGTGGCAACAGCGCACTG

GCACTGCAAACGGATGCACGTAATTCTGACCTGA

CCATTACGCAGCATGGTGGCGGTAACGGCGCTGA

TGTGGGCCAAGGTAGTGATGACAGCTCTATCGAC

CTGACCCAGCGCGGCTTTGGTAATAGTGCAACGCT

GGATCAATGGAACGGCAAAAATTCCGAAATGACC

GTTAAGCAGTTCGGCGGTGGCAATGGTGCGGCCG

TCGATCAAACCGCGTCCAACAGTTCCGTGAATGTT

ACGCAGGTTGGCTTTGGTAACAATGCAACCGCTC

ATCAATATAACGGCAAAAATGGATCCTCGGAACT

GAACATCTATCAGTACGGTGGCGGTAATTCAGCG

CTGGCCCTGCAAACCGATGCGCGTAACTCGGATCT

GACGATTACGCAGCATGGCGGTGGCAACGGTGCC

GATGTCGGTCAGGGCAGCGACGATTCTAGCATCG

ACCTGACGCAACGTGGCTTTGGTAACAGCGCCACC

This

work

115

CTGGACCAGTGGAATGGTAAAAATTCTGAAATGA

CCGTGAAGCAGTTCGGTGGCGGTAATGGTGCAGC

TGTTGATCAAACGGCAAGCAACAGTTCCGTCAAT

GTGACCCAAGTGGGCTTCGGTAACAATGCAACGG

CTCATCAATACAATGGTAAGAACGGATCCTCGGA

ACTGAACATTTACCAATACGGCGGTGGCAATAGT

GCCCTGGCGCTGCAAACCGATGCACGTAACTCCGA

TCTGACGATCACGCAGCACGGTGGCGGTAACGGT

GCTGATGTTGGCCAAGGTTCAGACGATTCTTCCA

TTGATCTGACGCAACGCGGCTTTGGTAACTCAGC

GACCCTGGACCAATGGAATGGTAAGAACTCGGAA

ATGACCGTCAAACAATTTGGCGGTGGCAACGGGG

CGGCCGTGGATCAAACCGCCTCTAACAGTTCCGTT

AATGTCACGCAAGTGGGTTTCGGCAATAACGCCA

CGGCCCACCAGTACAATGGTAAGAATGGATCCAG

CGAACTGAATATCTACCAATATGGTGGCGGTAAT

AGCGCCCTGGCTCTGCAGACGGATGCGCGTAACAG

CGATCTGACCATCACCCAGCACGGCGGTGGCAACG

GCGCAGACGTGGGTCAGGGCAGCGATGATTCTTC

TATCGACCTGACGCAGCGTGGTTTTGGTAACAGT

GCAACCCTGGATCAGTGGAATGGCAAGAACAGCG

AAATGACGGTTAAACAATTTGGTGGCGGTAACGG

GGCAGCTGTCGATCAAACGGCCAGTAATAGTTCC

GTGAACGTGACGCAAGTTGGTTTTGGCAATAACG

116

CGACCGCCCATCAATACAATGGCAAGAACGGATC

CAGCGAACTGAACATTTATCAATATGGCGGTGGC

AACTCTGCCCTGGCTCTGCAAACGGACGCCCGTAA

CTCGGACCTGACGATCACCCAACATGGTGGCGGTA

ATGGTGCCGACGTTGGCCAAGGTAGCGACGATTC

CAGCATTGATCTGACCCAACGTGGTTTCGGTAAC

AGCGCGACCCTGGATCAATGGAATGGCAAGAATA

GCGAAATGACGGTCAAACAATTCGGCGGTGGCAA

CGGAGCGGCCGTTGATCAAACCGCCAGTAACAGT

TCCGTCAACGTGACGCAAGTGGGTTTTGGCAATA

ATGCCACGGCCCACCAATACAATGGTAAAAACGG

ATCCTCTGAACTGAATATTTACCAGTATGGTGGC

GGTAACAGTGCCCTGGCACTGCAGACGGACGCGC

GTAACTCCGACCTGACGATCACGCAACATGGCGGT

GGCAATGGCGCCGATGTTGGCCAGGGTAGCGACG

ATAGTAGCATTGATTTAACCCAGCGTGGTTTCGG

CAATAGCGCCACGCTGGATCAGTGGAACGGTAAG

AATTCAGAAATGACGGTCAAGCAATTTGGTGGCG

GTAATGGGGCAGCTGTGGATCAGACGGCCAGCAA

TAGTTCCGTTAACGTCACCCAAGTGGGTTTCGGT

AACAACGCCACGGCTCATCAGTACAATGGTAAAA

ATGGATCCTCCGAGTTAAACATCTACCAGTATGG

CGGTGGCAATTCTGCCCTGGCCCTGCAAACGGACG

CGCGCAATAGCGATCTGACCATTACCCAACACGGT

117

GGCGGTAATGGCGCCGACGTGGGCCAGGGTAGCG

ATGATAGTTCCATTGACCTGACGCAACGCGGTTT

CGGCAACAGTGCGACGCTGGACCAATGGAACGGT

AAGAACTCTGAAATGACGGTGAAACAGTTTGGCG

GTGGCAATGGGGCGGCCGTCGATCAGACCGCGTCT

AACAGTTCCGTGAACGTGACACAGGTCGGTTTCG

GCAATAATGCCACCGCCCATCAGTACAATGGCAA

GAATGGATCCCAATACGGTGGCGGTAACTCGGCA

CTGGCACTGCAGACCGATGCACGTAATAGCGACCT

GACGATTACCCAACACGGCGGTGGCAATGGAGCA

GATGTGGGCCAGGGTTCTGATGACTCATCGATTG

ATCTGACGCAGCGTGGCTTCGGTAATTCAGCCACG

CTGGACCAGTGGAACGGTAAAAACAGCGAGATGA

CCGTTAAGCAATTCGGTGGCGGTAACGGAGCAGC

TGTTGATCAGACGGCGAGCAACAGCTCTGTCAAT

GTGACCCAGGTCGGTTTCGGTAATAATGCTACGG

CACATCAGTATCACCACCACCATCATCATCACTAA

gfp Fluorescent

reporter

ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTG

TCCCAATTCTTGTTGAATTAGATGGTGATGTTAA

TGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAA

GGTGATGCAACATACGGAAAACTTACCCTTAAAT

TTATTTGCACTACTGGAAAACTACCTGTTCCATG

GCCAACACTTGTCACTACTTTCGGTTATGGTGTTC

AATGCTTTGCGAGATACCCAGATCATATGAAACA

3

118

GCATGACTTTTTCAAGAGTGCCATGCCCGAAGGT

TATGTACAGGAAAGAACTATATTTTTCAAAGATG

ACGGGAACTACAAGACACGTGCTGAAGTCAAGTT

TGAAGGTGATACCCTTGTTAATAGAATCGAGTTA

AAAGGTATTGATTTTAAAGAAGATGGAAACATTC

TTGGACACAAATTGGAATACAACTATAACTCACA

CAATGTATACATCATGGCAGACAAACAAAAGAAT

GGAATCAAAGTTAACTTCAAAATTAGACACAACA

TTGAAGATGGAAGCGTTCAACTAGCAGACCATTA

TCAACAAAATACTCCAATTGGCGATGGCCCTGTCC

TTTTACCAGACAACCATTACCTGTCCACACAATCT

GCCCTTTCGAAAGATCCCAACGAAAAGAGAGACC

ACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGG

GATTACACATGGCATGGATGAACTATACAAATAA

mCherry Fluorescent

reporter

ATGGTGAGCAAGGGCGAAGAAGATAACATGGCCA

TCATCAAGGAGTTCATGCGCTTCAAGGTGCACAT

GGAGGGCTCCGTGAACGGCCACGAGTTCGAGATC

GAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCA

CCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGG

CCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTC

AGTTCATGTACGGCTCCAAGGCCTACGTGAAGCA

CCCCGCCGACATCCCCGACTACTTGAAGCTGTCCT

TCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAA

CTTCGAGGACGGCGGCGTGGTGACCGTGACCCAG

3

119

GACTCCTCCCTGCAGGACGGCGAGTTCATCTACAA

GGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACG

GCCCCGTAATGCAGAAGAAGACCATGGGCTGGGA

GGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCG

CCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCT

GAAGGACGGCGGCCACTACGACGCTGAGGTCAAG

ACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCC

CGGCGCCTACAACGTCAACATCAAGTTGGACATC

ACCTCCCACAACGAGGACTACACCATCGTGGAACA

GTACGAACGCGCCGAGGGCCGCCACTCCACCGGCG

GCATGGACGAGCTGTACAAGTAA

csgASpyTag Curliamyloid

material

subunit














This

work

120

AGTACGGCGGGGGCTCCGGCGGGGGCTCCGCGCAC

ATCGTTATGGTCGATGCATATAAACCCACCAAAT

AA

csgAFLAG Curliamyloid

material

subunit














AGTACGATTACAAGGATGACGATGACAAGTAA

This

work

csgAZnSpeptide Curliamyloid

material

subunit








This

work

121







AGTACGGTGGTGGTTCTTGCAACAACCCGATGCA

CCAGAACTGCTAA

csgAHisafterC‐

terminus

Curliamyloid

material

subunit














AGTACCACCATCACCATCACCACCATTAA

This

work

CCSpyCatche Gene

encodingthe

ATGTGTTGTTCGTACTACCATCACCATCACCATCA

CGATTACGACATCCCAACGACCGAAAACCTGTAT

5

122

r SpyCatcher

proteinwith

twoN‐

terminal

cysteinesfor

conjugation

toCdTe/CdS

QDs

TTTCAGGGCGCCATGGTTGATACCTTATCAGGTT

TATCAAGTGAGCAAGGTCAGTCCGGTGATATGAC

AATTGAAGAAGATAGTGCTACCCATATTAAATTC

TCAAAACGTGATGAGGACGGCAAAGAGTTAGCTG

GTGCAACTATGGAGTTGCGTGATTCATCTGGTAA

AACTATTAGTACATGGATTTCAGATGGACAAGTG

AAAGATTTCTACCTGTATCCAGGAAAATATACAT

TTGTCGAAACCGCAGCACCAGACGGTTATGAGGT

AGCAACTGCTATTACCTTTACAGTTAATGAGCAA

GGTCAGGTTACTGTAAATGGCAAAGCAACTAAAG

GTGACGCTCATATTTAA

csgAHisnoSS Gene

encodingthe

matureform

ofCsgA

protein

withoutthe

secretion

signal,witha

HistagatC‐

terminus;

purifiedasa

reference

standardfor

ATGGGTGTTGTTCCTCAGTACGGCGGCGGCGGTA

ACCACGGTGGTGGCGGTAATAATAGCGGCCCAAA

TTCTGAGCTGAACATTTACCAGTACGGTGGCGGT

AACTCTGCACTTGCTCTGCAAACTGATGCCCGTAA

CTCTGACTTGACTATTACCCAGCATGGCGGCGGTA

ATGGTGCAGATGTTGGTCAGGGCTCAGATGACAG

CTCAATCGATCTGACCCAACGTGGCTTCGGTAACA

GCGCTACTCTTGATCAGTGGAACGGCAAAAATTC

TGAAATGACGGTTAAACAGTTCGGTGGTGGCAAC

GGTGCTGCAGTTGACCAGACTGCATCTAACTCCTC

CGTCAACGTGACTCAGGTTGGCTTTGGTAACAAC

GCGACCGCTCATCAGTACCATCACCATCACCATCA

CCACTAA

22

123

quantitating

CsgA

productionin

biofilmsvia

dotblot

ampR AmpR ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCC

CTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC

ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA

AGATCAGTTGGGTGCACGAGTGGGTTACATCGAA

CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT

TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT

TTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC

GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGC

ATACACTATTCTCAGAATGACTTGGTTGAGTACT

CACCAGTCACAGAAAAGCATCTTACGGATGGCAT

GACAGTAAGAGAATTATGCAGTGCTGCCATAACC

ATGAGTGATAACACTGCGGCCAACTTACTTCTGA

CAACGATCGGAGGACCGAAGGAGCTAACCGCTTT

TTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATAC

CAAACGACGAGCGTGACACCACGATGCCTGTAGC

AATGGCAACAACGTTGCGCAAACTATTAACTGGC

GAACTACTTACTCTAGCTTCCCGGCAACAATTAAT

AGACTGGATGGAGGCGGATAAAGTTGCAGGACCA

4

124

CTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTAT

TGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT

CGCGGTATCATTGCAGCACTGGGGCCAGATGGTA

AGCCCTCCCGTATCGTAGTTATCTACACGACGGGG

AGTCAGGCAACTATGGATGAACGAAATAGACAGA

TCGCTGAGATAGGTGCCTCACTGATTAAGCATTG

GTAA

cmR CmR ATGGAGAAAAAAATCACTGGATATACCACCGTTG

ATATATCCCAATGGCATCGTAAAGAACATTTTGA

GGCATTTCAGTCAGTTGCTCAATGTACCTATAAC

CAGACCGTTCAGCTGGATATTACGGCCTTTTTAA

AGACCGTAAAGAAAAATAAGCACAAGTTTTATCC

GGCCTTTATTCACATTCTTGCCCGCCTGATGAATG

CTCATCCGGAATTCCGTATGGCAATGAAAGACGG

TGAGCTGGTGATATGGGATAGTGTTCACCCTTGT

TACACCGTTTTCCATGAGCAAACTGAAACGTTTT

CATCGCTCTGGAGTGAATACCACGACGATTTCCGG

CAGTTTCTACACATATATTCGCAAGATGTGGCGT

GTTACGGTGAAAACCTGGCCTATTTCCCTAAAGG

GTTTATTGAGAATATGTTTTTCGTCTCAGCCAAT

CCCTGGGTGAGTTTCACCAGTTTTGATTTAAACG

TGGCCAATATGGACAACTTCTTCGCCCCCGTTTTC

ACCATGGGCAAATATTATACGCAAGGCGACAAGG

TGCTGATGCCGCTGGCGATTCAGGTTCATCATGCC

4

125

GTCTGTGATGGCTTCCATGTCGGCAGAATGCTTA

ATGAATTACAACAGTACTGCGATGAGTGGCAGGG

CGGGGCGTAA

kanR KanR CTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTC

AAGAAGGCGATAGAAGGCGATGCGCTGCGAATCG

GGAGCGGCGATACCGTAAAGCACGAGGAAGCGGT

CAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCA

CGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGC

CACACCCAGCCGGCCACAGTCGATGAATCCAGAAA

AGCGGCCATTTTCCACCATGATATTCGGCAAGCA

GGCATCGCCATGGGTCACGACGAGATCCTCGCCGT

CGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCG

GCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATC

ATCCTGATCGACAAGACCGGCTTCCATCCGAGTAC

GTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCG

AATGGGCAGGTAGCCGGATCAAGCGTATGCAGCC

GCCGCATTGCATCAGCCATGATGGATACTTTCTCG

GCAGGAGCAAGGTGAGATGACAGGAGATCCTGCC

CCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCC

GCTTCAGTGACAACGTCGAGCACAGCTGCGCAAG

GAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCT

GCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAG

GTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGC

GCTGACAGCCGGAACACGGCGGCATCAGAGCAGC

4

126

CGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGC

CTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAA

TCCATCTTGTTCAATCATGCGAAACGATCCTCATC

CTGTCTCTTGATCAGATCTTGATCCCCTGCGCCAT

CAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTA

CTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCC

CCAGCTGGCAATTCCG

specR SpecR ATGCGCTCACGCAACTGGTCCAGAACCTTGACCGA

ACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTC

ATGGCTTGTTATGACTGTTTTTTTGGGGTACAGT

CTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTAC

GCCGTGGGTCGATGTTTGATGTTATGGAGCAGCA

ACGATGTTACGCAGCAGGGCAGTCGCCCTAAAAC

AAAGTTAAACATCATGAGGGAAGCGGTGATCGCC

GAAGTATCGACTCAACTATCAGAGGTAGTTGGCG

TCATCGAGCGCCATCTCGAACCGACGTTGCTGGCC

GTACATTTGTACGGCTCCGCAGTGGATGGCGGCCT

GAAGCCACACAGTGATATTGATTTGCTGGTTACG

GTGACCGTAAGGCTTGATGAAACAACGCGGCGAG

CTTTGATCAACGACCTTTTGGAAACTTCGGCTTCC

CCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAG

TCACCATTGTTGTGCACGACGACATCATTCCGTGG

CGTTATCCAGCTAAGCGCGAACTGCAATTTGGAG

AATGGCAGCGCAATGACATTCTTGCAGGTATCTT

4

127

CGAGCCAGCCACGATCGACATTGATCTGGCTATCT

TGCTGACAAAAGCAAGAGAACATAGCGTTGCCTT

GGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCG

GTTCCTGAACAGGATCTATTTGAGGCGCTAAATG

AAACCTTAACGCTATGGAACTCGCCGCCCGACTGG

GCTGGCGATGAGCGAAATGTAGTGCTTACGTTGT

CCCGCATTTGGTACAGCGCAGTAACCGGCAAAAT

CGCGCCGAAGGAGGTCGCTGCCGACTGGGCAATG

GAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACG

TGAAGCTAGACAGGCTTATCTTGGACAAGAAGAA

GATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAG

AATTTGTCCACTACGTGAAAGGCGAGATCACCAA

GGTAGTCGGCAAATAA

128

SupplementaryTableA‐2|Plasmidsusedinthiswork.

Plasmidname PlasmidID Description Source

pE‐AmpR‐p(lac)‐

luxI

Designated“pSND‐

1”in27

pSND‐1 ColE1origin,Ampresistance,p(lac)

promoter,luxIoutputgene

27

pZA‐AmpR‐

pL(tetO)‐gfp

Designated

“pZA11G”in4

pZA11G p15Aorigin,Ampresistance,pL(tetO)

promoter,gfpoutputgene

4

pZS‐CmR‐pL(lacO)‐

gfp

Designated

“pZS32G”in4

pZS32G pSC101origin,Cmresistance,

pL(lacO)promoter,gfpoutputgene

4

pZE‐AmpR‐rr12‐

pL(tetO)‐gfp

rrjt12(11)g ColE1origin,Ampresistance,rr12

riboregulator,pL(tetO)promoter,gfp

outputgene

3

129

Designated

“rrjt12(11)g”in3

pZE‐KanR‐rr12y‐

pLuxR‐gfp

Designated

“rr12y(rii)g”in3

rr12y(rii)g ColE1origin,Kanresistance,rr12y

riboregulator,pLuxRpromoter,gfp

outputgene

3

pZE‐KanR‐rr10‐

pL(lacO)‐mCherry

Designated

“rrjc10(22)mc”in3

rrjc10(22)mc ColE1origin,Kanresistance,rr10

riboregulator,pL(lacO)promoter,

mCherryoutputgene

3

pZE‐AmpR‐pLuxR‐

lacZalpha

pAYC001 ColE1origin,Ampresistance,pLuxR

promoter,lacZalphaoutputgene

Giftof

Jacob

Rubens,

LuLab

pZA‐CmR‐rr12‐

pL(tetO)‐csgA

pAYC002 p15Aorigin,Cmresistance,rr12

riboregulator,pL(tetO)promoter,

csgAoutputgene

This

work

130

pZA‐CmR‐rr12‐

pL(tetO)‐csgAHis



csgAHisoutputgene

This

work

pZA‐CmR‐rr12‐

pL(tetO)‐mCherry



mCherryoutputgene

This

work

pZA‐CmR‐rr12y‐

pLuxR‐8XcsgAHis

pAYC005 p15Aorigin,Cmresistance,rr12y

riboregulator,pLuxRpromoter,

8XcsgAHisoutputgene

This

work


pLuxR‐csgA


riboregulator,pLuxRpromoter,csgA

outputgene

This

work


pLuxR‐csgAHis



csgAHisoutputgene

This

work


pLuxR‐gfp


riboregulator,pLuxRpromoter,gfp

outputgene

This

work

pZS‐AmpR‐rr10‐

pL(lacO)‐lacZalpha

pAYC009 pSC101origin,Cmresistance,rr12y

riboregulator,pL(lacO)promoter,

lacZalphaoutputgene

This

work

131

pZE‐AmpR‐proB‐

mCherry

pAYC010 ColE1origin,Ampresistance,proB

promoter,mCherryoutputgene

Giftof

TomiJun,

LuLab


pLuxR‐csgASpyTag



csgASpyTagoutputgene

This

work

pZA‐CmR‐rr12‐

pL(tetO)‐csgAFLAG



csgAFLAGoutputgene

This

work


csgAZnSpeptide

pAYC013 pSC101origin,Cmresistance,

pL(lacO)promoter,csgAZnSpeptide

outputgene

This

work


csgA


pL(lacO)promoter,csgAZnSoutput

gene

This

work


csgAHisafterC‐terminus


pL(lacO)promoter,csgAHisafterR5

outputgene

This

work

pDEST14‐T7‐

CCSpyCatcher

pAYC016 pBR322origin,Ampresistance,T7

promoter,CCSpyCatcheroutputgene

This

work

pET11d‐T7‐ pAYC017 pBR322origin,Ampresistance,T7 Giftof

Chao

132

csgAHisnoSS promoter,csgAHisnoSS outputgene Zhong,Lu

Lab

pZS4Int‐lacI/tetR pAYC018 pSC101origin,PROcassette

(Placiq/lacI,PN25/tetR,SpecR)

Expressys

133

SupplementaryTableA‐3|Cellstrainsusedinthiswork.

Strainname StrainID Description Antibiotic

resistance*

Source

MG1655ompR234

Referredtoas

“ompR234”infigures

MG1655

ompR234

E.coli strainwith

ompR234mutationthat

confersabilityto

producecurlifibrilsin

liquidM63minimal

media.

Kan 9

BW25113

∆csgA::aph

BW25113

∆csgA::aph

E.coli strainwithcsgA

knock‐outachievedby

replacementwith

kanamycinresistance

cassette(aph).

Kan 6

BW25113∆csgA fAYC001 E.coli strainwithcsgA

knockedout

N/A Thiswork

BL21DE3pLysS BL21DE3

pLysS

E.colistrainforinducible

proteinexpression

Cm Stratagene

MG1655PRO∆csgA

ompR234

Referredtoas“∆csgA

fAYC002 E.coli strainwith

constitutivehighlevel

expressionoftetRand

Spec,Kan Thiswork

* Kanamycin (Kan), Spectinomycin (Spec), Chloramphenicol (Cm), Ampicillin (Amp)

134

ompR234”infigures lacI fromPRO cassette

derivedfrompZS4Int‐

lacI/tetR,withcsgA

knockedout,andwith

ompR234mutationthat

confersabilityto

producecurlifibrilsin

liquidM63minimal

media.

aTcReceiver/CsgAHis fAYC003 E.coli strainthat

expressesCsgAHisunder

tightregulationbyan

anhydrotetracycline

(aTc)inducer‐

responsive

riboregulator.Madeby

transformingpZA‐CmR‐

rr12‐pL(tetO)‐csgAHis

plasmidintoMG1655

PRO∆csgAompR234.

Spec, Kan,

Cm

Thiswork

AHLReceiver/CsgA fAYC004 E.coli strainthat

expressesCsgAunder

tightregulationbyan

acyl‐homoserinelactone

Spec,Kan,

Cm

Thiswork

135

(AHL)inducer‐

responsive



rr12y‐pLuxR‐csgA

plasmidintoMG1655

PRO∆csgAompR234.

AHLSender+

aTcReceiver/CsgA

fAYC005 E.coli strainthat

constitutivelyproduces

AHLatalowbasallevel

andinduciblyexpresses

CsgAundertight

regulationbyanaTc

inducer‐responsive


co‐transformingpZA‐

CmR‐rr12‐pL(tetO)‐csgA

andpE‐AmpR‐p(lac)‐luxI

intoMG1655PRO∆csgA

ompR234.

Spec,Kan,

Cm,Amp

Thiswork

AHLReceiver/CsgAHis fAYC006 E.coli strainthat


tightregulationbyan

AHLinducer‐responsive

Spec,Kan,

Cm,Amp

Thiswork

136


co‐transformingpZA‐

CmR‐rr12y‐pLuxR‐

csgAHisandpZS‐AmpR‐

rr10‐pL(lacO)‐lacZalpha


ompR234.

AHLReceiver/8XCsgAHis fAYC007 E.coli strainthat

expresses8XCsgAHis

undertightregulationby

anAHLinducer‐

responsive



rr12y‐pLuxR‐8XcsgAHis


ompR234.

Spec,Kan,

Cm

Thiswork

aTcReceiver/CsgA fAYC008 E.coli strainthat

expressesCsgAunder

tightregulationbyan

anhydrotetracycline

(aTc)inducer‐

responsive


Spec,Kan,

Cm

Thiswork

137


rr12‐pL(tetO)‐csgA

plasmidintoMG1655

PRO∆csgAompR234.

aTcReceiver/mCherry fAYC009 E.coli strainthat

expressesmCherry


ananhydrotetracycline

(aTc)inducer‐

responsive



rr12‐pL(tetO)‐mCherry

plasmidintoMG1655

PRO∆csgAompR234.

Spec,Kan,

Cm

Thiswork

AHLReceiver/GFP fAYC010 E.coli strainthat

expressesGFPunder

tightregulationbyan

acyl‐homoserinelactone

(AHL)inducer‐

responsive



rr12y‐pLuxR‐gfp

Spec,Kan,

Cm

Thiswork

138

plasmidintoMG1655

PRO∆csgAompR234.

mCherry+

aTcReceiver/CsgAHis



tightregulationbyan

anhydrotetracycline

(aTc)inducer‐

responsive

riboregulator,and

constitutivelyexpresses

mCherry.Madebyco‐

transformingpZE‐

AmpR‐proB‐mCherry

andpZA‐CmR‐rr12‐

pL(tetO)‐csgAHisinto

MG1655PRO∆csgA

ompR234.

Spec,Kan,

Cm,Amp

Thiswork

MG1655ompR234/

pZE‐AmpR‐proB‐

mCherry


expressesCsgAunder

controlofnativecurli

operon,and


mCherry.Madeby

transformingpZE‐

Spec,Kan,

Amp

Thiswork

139


plasmidintoMG1655

ompR234.

MG1655PRO csgA

ompR234/pZE‐




mCherry.Madeby

transformingpZE‐


plasmidintoMG1655

PRO∆csgAompR234.

Spec,Kan,

Amp

Thiswork

AHLReceiver/CsgASpyTag fAYC014 E.coli strainthat

expressesCsgASpyTag


anAHLinducer‐

responsive



rr12y‐pLuxR‐csgASpyTag


ompR234.

Spec,Kan,

Cm

Thiswork

aTcReceiver/CsgAFLAG fAYC015 E.coli strainthat

expressesCsgAFLAG


Spec,Kan,

Cm

Thiswork

140

ananhydrotetracycline

(aTc)inducer‐

responsive



rr12‐pL(tetO)‐csgAFLAG

plasmidintoMG1655

PRO∆csgAompR234.

BL21DE3pLysS/

pDEST14‐T7‐

CCSpyCatcher


expressesCCSpyCatcher

wheninducedbyIPTG.

Madebytransforming

pDEST14‐T7‐

CCSpyCatcherintoBL21

DE3pLysS.

Cm,Amp Thiswork

BL21T7ExpressIq/

pET11d‐T7‐

csgAHisnoSS


expressesCsgAHisnoSS

wheninducedbyIPTG.

Madebytransforming

pET11d‐T7‐csgAHisnoSS

intoBL21T7ExpressIq.

Cm,Amp Giftof

Chao

Zhong,Lu

Lab

BW25113∆csgA/


fAYC018 E.colistrainthat

expressesCsgAunder

Kan,Cm,

Spec

Thiswork

141

csgA+pZS4Int‐

lacI/tetR

controlofpL(lacO)

promoter,alongwith

constitutiveexpression

oflacIandtetR.Madeby

co‐transformingpZS‐

CmR‐pL(lacO)‐csgAand

pZS4Int‐lacI/tetRinto

BW25113∆csgA.

BW25113∆csgA/


csgAZnSpeptide(also

referredtoas

“ csgA/CsgAZnS

peptide”)


expressesCsgAZnSpeptide

undercontrolof

pL(lacO)promoter.

Madebytransforming


csgAZnSpeptideinto

BW25113∆csgA.

Kan,Cm Thiswork

142

BW25113∆csgA/


csgA(alsoreferred

toas“ csgA/CsgA”)


expressesCsgAunder

controlofpL(lacO)

promoter.Madeby

transformingpZS‐CmR‐

pL(lacO)‐csgAinto

BW25113∆csgA.

Kan,Cm Thiswork

BW25113∆csgA/


csgAHisafterC‐terminus


expressesCsgAHisafterR5

undercontrolof

pL(lacO)promoter.

Madebytransforming


csgAHisafterR5into

BW25113∆csgA.

Kan,Cm Thiswork

143

References

1 Chapman,M.R.etal.RoleofEscherichiacolicurlioperonsindirectingamyloidfiberformation.Science295,851‐855,doi:10.1126/science.1067484295/5556/851[pii](2002).

2 Sambrook,J.,Fritsch,E.F.&Maniatis,T.Molecularcloning:alaboratorymanual.,(ColdSpringLaboratoryPress2,1989).

3 Callura,J.M.,Cantor,C.R.&Collins,J.J.Geneticswitchboardforsyntheticbiology

applications.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica109,5850‐5855,doi:10.1073/pnas.1203808109(2012).

4 Lutz,R.&Bujard,H.Independentandtightregulationoftranscriptionalunitsin

EscherichiacoliviatheLacR/O,theTetR/OandAraC/I1‐I2regulatoryelements.NucleicAcidsRes25,1203‐1210,doi:gka167[pii](1997).

5 Zakeri,B.etal.Peptidetagformingarapidcovalentbondtoaprotein,through

engineeringabacterialadhesin.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica109,E690‐697,doi:10.1073/pnas.1115485109(2012).

6 Baba,T.etal.ConstructionofEscherichiacoliK‐12in‐frame,single‐gene

knockoutmutants:theKeiocollection.MolSystBiol2,20060008,doi:msb4100050[pii]10.1038/msb4100050(2006).

7 Datsenko,K.A.&Wanner,B.L.One‐stepinactivationofchromosomalgenesinEscherichiacoliK‐12usingPCRproducts.ProcNatlAcadSciUSA97,6640‐6645,doi:10.1073/pnas.120163297120163297[pii](2000).

8 Cherepanov,P.P.&Wackernagel,W.GenedisruptioninEscherichiacoli:TcRandKmRcassetteswiththeoptionofFlp‐catalyzedexcisionoftheantibiotic‐resistancedeterminant.Gene158,9‐14(1995).

9 Prigent‐Combaret,C.etal.Complexregulatorynetworkcontrolsinitialadhesion

andbiofilmformationinEscherichiacoliviaregulationofthecsgDgene.Journalofbacteriology183,7213‐7223(2001).

10 Prigent‐Combaret,C.etal.Developmentalpathwayforbiofilmformationincurli‐

producingEscherichiacolistrains:roleofflagella,curliandcolanicacid.Environmentalmicrobiology2,450‐464(2000).

11 Schneider,C.A.,Rasband,W.S.&Eliceiri,K.W.NIHImagetoImageJ:25yearsof

imageanalysis.Naturemethods9,671‐675(2012).12 Heydorn,A.etal.Quantificationofbiofilmstructuresbythenovelcomputer

programCOMSTAT.Microbiology146(Pt10),2395‐2407(2000).

144

13 Collinson,S.K.,Emody,L.,Muller,K.H.,Trust,T.J.&Kay,W.W.Purificationandcharacterizationofthin,aggregativefimbriaefromSalmonellaenteritidis.Journalofbacteriology173,4773‐4781(1991).

14 Hung,C.etal.Escherichiacolibiofilmshaveanorganizedandcomplex

extracellularmatrixstructure.mBio4,e00645‐00613,doi:10.1128/mBio.00645‐13(2013).

15 Wang,X.,Hammer,N.D.&Chapman,M.R.Themolecularbasisoffunctional

bacterialamyloidpolymerizationandnucleation.TheJournalofbiologicalchemistry283,21530‐21539,doi:10.1074/jbc.M800466200(2008).

16 O'Toole,G.A.Microtiterdishbiofilmformationassay.Journalofvisualized

experiments:JoVE,doi:10.3791/2437(2011).17 Zhou,Y.,Smith,D.R.,Hufnagel,D.A.&Chapman,M.R.Experimental

manipulationofthemicrobialfunctionalamyloidcalledcurli.MethodsMolBiol966,53‐75,doi:10.1007/978‐1‐62703‐245‐2_4(2013).

18 Ishiguro,E.E.,Ainsworth,T.,Trust,T.J.&Kay,W.W.Congoredagar,a

differentialmediumforAeromonassalmonicida,detectsthepresenceofthecellsurfaceproteinarrayinvolvedinvirulence.Journalofbacteriology164,1233‐1237(1985).

19 Weinberg,E.D.Double‐gradientagarplates.Science125,196(1957).20 Deng,Z.etal.AqueoussynthesisofzincblendeCdTe/CdSmagic‐core/thick‐shell

tetrahedral‐shapednanocrystalswithemissiontunabletonear‐infrared.JournaloftheAmericanChemicalSociety132,5592‐5593,doi:10.1021/ja101476b(2010).

21 Mao,C.etal.Viralassemblyoforientedquantumdotnanowires.Proceedingsof

theNationalAcademyofSciencesoftheUnitedStatesofAmerica100,6946‐6951,doi:10.1073/pnas.0832310100(2003).

22 Zhong,C.,Gurry,T.,Cheng,A.,Downey,J.,Stultz,C.M.&Lu,T.K.Biologically

InspiredEngineeringofStrong,Self‐Assembling,andMulti‐FunctionalUnderwaterAdhesiveswithSyntheticBiology.Inreview.

23 Wang,X.,Smith,D.R.,Jones,J.W.&Chapman,M.R.Invitropolymerizationofa

functionalEscherichiacoliamyloidprotein.TheJournalofbiologicalchemistry282,3713‐3719,doi:10.1074/jbc.M609228200(2007).

24 Romero,D.,Aguilar,C.,Losick,R.&Kolter,R.Amyloidfibersprovidestructural

integritytoBacillussubtilisbiofilms.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica107,2230‐2234,doi:10.1073/pnas.0910560107(2010).

145

25 Bieschke,J.etal.Small‐moleculeconversionoftoxicoligomerstonontoxicbeta‐sheet‐richamyloidfibrils.Naturechemicalbiology8,93‐101,doi:10.1038/nchembio.719(2012).

26 Robinson,L.S.,Ashman,E.M.,Hultgren,S.J.&Chapman,M.R.Secretionofcurli

fibresubunitsismediatedbytheoutermembrane‐localizedCsgGprotein.Molecularmicrobiology59,870‐881,doi:10.1111/j.1365‐2958.2005.04997.x(2006).

27 Basu,S.,Gerchman,Y.,Collins,C.H.,Arnold,F.H.&Weiss,R.Asynthetic

multicellularsystemforprogrammedpatternformation.Nature434,1130‐1134,doi:nature03461[pii]10.1038/nature03461(2005).

28 Schweizer,H.P.&Hoang,T.T.AnimprovedsystemforgenereplacementandxylEfusionanalysisinPseudomonasaeruginosa.Gene158,15‐22(1995).

Engineered Biofilms for Materials Production and Patterning

Documents

Transcript of Engineered Biofilms for Materials Production and Patterning