178D.Woloznyetal./Engineering5(2019)173–180Fig.6.Demonstrationofdeviceuse.(a)Agarisaddedtothebacterialwellwithinthebioassaydevice.(b)Thedevicelidisalignedwiththedevicebaseandpressedwiththethumbuntilthepress-?tseals.Usingthethumb,forcecanbeappliedtothelid,whichreleasesthepress-?tanddirectsforceontothestress-focusingcutout.(c)Thebacterialwellthenfallsintothesample,whereitinoculatesthesamplewiththebiosensor.(d)Theliquidandbacteriawithinthetesttuberemainisolatedfromtheenvironment.Fig.7.Biosensordose–responsecurve.(a)Wheninducedwith3O-C12,thebiosensorgoesfromanun-inducedstatetoaninducedstate.Afterbeinginduced,thebiosensor?uorescesbyproducingmCherryprotein.Thebiosensorwasexposedtovaryingconcentrationdecadesof3O-C12andmaintainedinexponentialstate.Datawerecollectedfor10000eventsfortheun-inducedandthe100nmoláLà13O-C12samples(threesamples).(b)After8hofinduction,thebiosensorreachesanequilibriumstateofmCherryexpression,whichcanbequanti?ed.Thesystemcanthenbe?ttedtoaHillfunctionwithanR2valueof0.987.(c)Withenoughtime,thesensorbecomesatoggleswitchandisabletooutputanon/offresponse.useoflivingbiosensors,whichcanpotentiallybeintegratedinparallelwithcell-freedetectionmethodsinthefuture.Weenvisionoursystembeingespeciallybene?cialinareasthatlacksigni?cantmedicalinfrastructureoraccesstohealthcare.The3O-C12biosensorisabletodetect3O-C12concentrationsrangingfrom0.1to10lmoláLà1.Itiswelldocumented[36–39]thataP.aeruginosainfectionintherespiratorytractscancausetissuein?ammation.Althoughthespeci?cmethodofactionofchemical3O-C12remainsunknown,itisbelievedtooccurthroughinteractionswithtoll-likereceptorsonthecell’ssurface[40],whichproduceanin?ammatoryresponse.Thisin?ammationistriggeredbya3O-C12concentrationintherangefrom10to100lmoláLà1.Therangeofconcentrationsfoundininfectedlungsliesoutsideofthedynamicresponserangeofour3O-C12biosensor.However,astraightforwardseriesofdilutions(e.g.,1/2,1/4,...)withmedia—asisdoneforotherquanti?cationassays,suchastheBradfordAssay[41]—canbeperformedbeforeanalysis.Dilutingthesputumcollectedinmediawillalsoallowthecellstodividefasterwithamoresuitableenvironmentforgrowth.Thebioassaydevicecanbefabricatedanywherea3Dprinterisavailable.The3Dprinterthatweusedforthisresearchissoldforlessthan2000USD,andmanynew3Dprinterscanbepurchasedforpricesrangingfrom200to500USD.Ourbioassaydevicecanalsobefabricatedquickly,asneeded,withdevicesreadyafterlessthan2h(Fig.8(a)).Thematerialcosttomanufactureoneofthe3D-printeddevicesdescribedhereisapproximately0.50USD,using8gofABS.Incomparisonwiththebiosensorhousingandcover,thebiosensoritselfcontributesonlyasmallportionoftheoverallcostofthedevice.Theprintercanbeusedatanylocation,makingthedeploymentofthisdevice?exible.Biosensorscanalsobereadilygrown,freeze-dried,andshippedworldwide.Withasmallquantityoffreeze-driedbiosensorloadedintothedevices(Fig.8(b)),theycanbesealedandbereadyforuse.Thedevicescanthenbedirectlytransportedtothepoint-of-careforuse(Fig.8(c)).Inthecaseofanaccident,inwhichadevicecomesintocontactwiththeenvironment,itisimportanttoquantifyhowwellthedeviceisolatesthebiosensorfromtheenvironment.Anexperimentusingnutrient-richmedia(Fig.8(d))wasusedtosimulateaworst-casescenario.Whenshakenat140ráminà1in100mLofmedia(Fig.8(e)),thebacterialwellthathadbeenintroducedintothemediawasabletopropagatecellsimmediately.Whentheentireintactdevicewassubmergedinmedia,thesealeddevicewasabletoisolatethebiosensorfromtheenvironmentforaperiodof14hbeforethecellsescapedandshowedvisibleturbidityin100mLofmedia.Inthe?eld,asputumsample(Fig.9(a))canbecollectedfromthepatientanddilutedintoasampletubewithcellmedia.Toensurethatnobacteriaoutcompetethekanamycin-tolerantlivingbiosensorinthesample,akanamycinantibioticcouldbeadded,makingthebacterialsamplesfromthepatient’ssputumunableD.Woloznyetal./Engineering5(2019)173–180179Fig.8.Fromtheprinter,tothepatient,tothelab.(a)A3Dprinterisusedtoprintthebioassaydevice.(b)Thegeneticallyengineeredsensorisculturedwithinthe3D-printedbioassaydevice.(c)Thedevicewiththesensoristransportedtothepatient’slocation.(d)Thedeviceiswater-resistant,sointheeventthatthedevicefallsintoaliquid,thereistimetoremoveitwithouttheescapeofGMOs.(e)Whenthedeviceisusednormally,andthebacteriaaredirectlydepositedintonutrient-richmedia,theywillimmediatelybegintogrow.However,thesealeddevicecouldalsopreventliquidfromreachingthecellsforaperiodof14hintheeventofdevicemishandlingresultinginemersioninenvironmentalliquids.Datacollectedfor10000eventsforthesealedandunsealedthreesamples.tosurvive.Thebiosensorcoverwouldthenbepressed?rmlyandactuated,forcingthefreeze-driedbacterialpellettoenterthesputumsample.Next,thetubecouldbeincubatedandanalyzedinthe?eld(Fig.9(b)).Thebiosensorcansimultaneouslyquantifyspeci?clevelsof3O-C12exposurewhenmaintainedinexponentialstateorcanswitchintoanon/offbehaviorbysimplyaddingthebiosensortothesample(Fig.9(c)).A9hincubationallowsthebiosensortoturntheliquidsamplevisiblyred,thusservingasatwo-statesystemthatisusefultovisuallydetermineinthe?eldwhetherapatienthasaP.aeruginosainfection.Thedevicewasdesignedtobefabricatedandusedbypersonnelorvolunteerswithminimaltraining.Afterattachmenttoaculturetube,thedeviceandculturetubecanbegrippedbythehand,andthethumbcanbeusedinalateralpinchgriptoactivatethesensor.Theaveragelateralpinchstrengthofanadultusingthedominanthandrangesfromapproximately5.0–7.5kgofforce[42],whichissuf?cientfortheuseofthisdevice.Duetothedesignofthestress-focusingcutoutpresentedhere,thelateralpinchforcegeneratedbyanadultwillallowthedevicetoreliablyfunctioninthe?eld.5.ConclusionTheexperimentalresultspresentedhereindicatethatthe3O-C12biosensorisabletosenseinducerconcentrationswhenP.aeruginosabio?lmsarepresent,asinthecaseofrecurringinfections.Thelivingbiosensor,whileusefulforthepurposeofdetection,hasthepotentialtofacefewerregulatoryhurdlesduetoitsenclosurewithinthebioassaydevice,whichpermitsitscon-taineddeploymentoutsidethelab.The3D-printeddeviceturnstheGMOintoacomponentoftheoveralldeviceandensuresthatthebiosensorisisolatedfromtheenvironment.Thus,3Dprintingthisdeviceenablesourlivingbiosensor,withitsversatilitytoFig.9.Biosensoractivityoncontactwith3O-C12.(a)Asputumsampleistakenfromthepatientandthe3D-printeddeviceiscollapsedtoreleasethebacteriaintothesample.(b)Thebiosensorisbuilttoenablebothquanti?cationof3O-C12concentrationandamoresimpleon/offbehaviorinwhich3O-C12atorabove10nmoláLà1willturnthesamplevisiblyred.(c)Thebiosensorturnsvisiblyredwitha98-fold?uorescenceincrease,comparedwiththeinitial?uorescencevalue,F/F0,after10hofexposuretotheinducer.mCherryproteinproductioncontinuesaslongascellsremainexposedtotheinducer.Datacollectedfor10000eventsfortheun-inducedandthe100nmoláLà13O-C12samples(threesamples).180D.Woloznyetal./Engineering5(2019)173–180processenvironmentalinput,tobe?eld-deployedasanovel,cost-effectivediagnostictechnology.AcknowledgementsTheauthorsacknowledgesupportfromfundingfromfederalagenciesoftheUnitedStatesincluding,theNationalScienceFoundation(1709238),aswellasfundingfromOf?ceofNavalResearch(N00014-17-12306andN00014-15-1-2502),andtheAirForceOf?ceofScienti?cResearch(FA9550-13-1-0108).Nofundingagenciesplayedasigni?cantroleinthestudydesign.AuthorcontributionsWoloznyandRuderconceivedtheideaforthesystemdescribedhere.Allauthorsdesignedandperformedtheexperiments,analyzedthedata,discussedtheresults,wrotethismanuscriptandcommentedonthepaper.Wolozny,Lake,Long,andRuderrevisedthemanuscript.CompliancewithethicsguidelinesDanielWolozny,JohnR.Lake,PaulG.Movizzo,ZhichengLong,andWarrenC.Ruderdeclarethattheyhavenocon?ictofinterestor?nancialcon?ictstodisclose.References[1]GardnerTS,CantorCR,CollinsJJ.ConstructionofagenetictoggleswitchinEscherichiacoli.Nature2000;403(6767):339–42.[2]ElowitzMB,LeiblerS.Asyntheticoscillatorynetworkoftranscriptionalregulators.Nature2000;403(6767):335–8.[3]BalagaddéFK,SongH,OzakiJ,CollinsCH,BarnetM,ArnoldFH,etal.AsyntheticEscherichiacolipredator-preyecosystem.MolSystBiol2008;4(1):187.[4]LitcofskyKD,AfeyanRB,KromRJ,KhalilAS,CollinsJJ.Iterativeplug-and-playmethodologyforconstructingandmodifyingsyntheticgenenetworks.NatMethods2012;9(11):1077–80.[5]BrennerK,KarigDK,WeissR,ArnoldFH.Engineeredbidirectionalcommunicationmediatesaconsensusinamicrobialbio?lmconsortium.ProcNatlAcadSciUSA2007;104(44):17300–4.[6]TamsirA,TaborJJ,VoigtCA.RobustmulticellularcomputingusinggeneticallyencodedNORgatesandchemical‘wires’.Nature2011;469(7329):212–5.[7]McDanielLE,BaileyEG,ZimmerliA.Effectofoxygen-supplyratesongrowthofEscherichiacoli.I.Studiesinunbaf?edandbaf?edshake?asks.ApplMicrobiol1965;13:109–14.[8]RatkowskyDA,OlleyJ,McMeekinTA,BallA.Relationshipbetweentemperatureandgrowthrateofbacterialcultures.JBacteriol1982;149(1):1–5.[9]KuzmaJ,BesleyJC.Ethicsofriskanalysisandregulatoryreview:frombio-tonanotechnology.NanoEthics2008;2(2):149–62.[10]GregorowiusD,Lindemann-MatthiesP,HuppenbauerM.Ethicaldiscourseontheuseofgeneticallymodi?edcrops:areviewofacademicpublicationsinthe?eldsofecologyandenvironmentalethics.JAgricEnvironEthics2012;25(3):265–93.[11]KilamaWL.Healthresearchethicsinpublichealth:trialsandimplementationofmalariamosquitocontrolstrategies.ActaTrop2009;112(Suppl1):S37–47.[12]SchmidtM.Specialissue:societalaspectsofsyntheticbiology.SystSynthBiol2009;3(1–4):1–2.[13]KuzmaJ,TanjiT.Unpackagingsyntheticbiology:identi?cationofoversightpolicyproblemsandoptions.RegulGovernance2010;4(1):92–112.[14]MelchelsFPW,FeijenJ,GrijpmaDW.Areviewonstereolithographyanditsapplicationsinbiomedicalengineering.Biomaterials2010;31(24):6121–30.[15]DimitrovD,SchreveK,DeBeerN.Advancesinthreedimensionalprinting—stateoftheartandfutureperspectives.RapidPrototypingJ2006;12(3):136–47.[16]SchubertC,VanLangeveldMC,DonosoLA.Innovationsin3Dprinting:a3Doverviewfromopticstoorgans.BrJOphthalmol2014;98(2):159–61.[17]VentolaCL.Medicalapplicationsfor3Dprinting:currentandprojecteduses.PT2014;39(10):704–11.[18]GambelloMJ,IglewskiBH.CloningandcharacterizationofthePseudomonasaeruginosalasRgene,atranscriptionalactivatorofelastaseexpression.JBacteriol1991;173(9):3000–9.[19]KiratisinP,TuckerKD,PassadorL.LasR,atranscriptionalactivatorofPseudomonasaeruginosavirulen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2 (2016) xxx–xxxContents lists available at ScienceDirect
Engineering
ResearchSynthetic Biology—Article一种可实现合成生物传感器现场部署的增材制造方法abDaniel Wolozny a, John R. Lake b, Paul G. Movizzo b, Zhicheng Long b,*, Warren C. Ruder b,* Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USAa r t i c l e i n f oArticle history:Received 13 May 2018Revised 25 September 2018Accepted 17 December 2018Available online 22 December 2018摘要合成生物学工具可用于设计活体生物传感器，报告目标分析物的存在。虽然这些工程细胞生物传感器在实验室外具有许多潜在应用，但由于它们属于转基因生物（genetically modified organism， GMO），通常被认为具有危险性。因此，如何在实验室外使用转基因生物的同时，降低将其释放到环境中的风险就至关重要。本文描述了一种包含合成生物电路的生物传感系统。含有该系统的大肠杆菌（Escherichia coli）被置于一个特制的 3D 打印的试管盖内。这些转基因生物能够检测到一种条件致病菌铜绿假单胞菌（Pseudomonas aeruginosa）的化学群体信号。在该设备中，活体生物传感器可以在不接触环境的情况下，接触感兴趣的样本。细胞可以在培养管内进行现场可视化分析，也可以送回实验室进行进一步分析。许多生物传感器缺乏现场部署所需的多功能性，由于缺乏资源和装置，许多疾病可能无法诊断。我们的生物检测设备利用3D 打印技术，为现场部署活体生物传感器制造了一种便携式、模块化和廉价的设备。
? 2019 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher
Education Press Limited Company This is an open access article under the CC BY-NC-ND license
关键词合成生物学增材制造生物传感器1.引言合成生物学是生物学和工程学的结合，它试图通过应用工程学原理来设计细胞控制的遗传元件，以此获得对自然界的新认知和利用。合成生物学常见例子包括记忆开关[1]和振荡器[2]电路，它们可以使细胞根据化学或其他信号的输入，在不同的状态之间进行切换。这种感知环境输入的能力使细胞能够充当生物传感器。合成工程生物传感器[3–6]利用活细胞的多功能性，可以实现对输入信号的检测和对输出信号的分析。活细胞，尤其是细菌，可以在许多条件下存活[7,8]，并且可以被设计成在临床环境中检测和诊断感染性的生物标志物。转基因生物（genetically modified organism，GMO）受到诸多法律约束和政府机构的监管[9–13]，这使得它们难以在实验室外进行应用。为了使工程生物传感器能够用于现场疾病诊断，必须满足相关规定。增材制造（俗称3D打印）采用三维打印的模式，它可以作为一种解决方案，使转基因生物在实验室外使用时与环境隔离。30年前，核心增材制造方法首次实现商业化[14]。此后，增材制造技术发展迅速，3D打印机得到广泛应用。利用3D打印机连续沉积挤压出细丝层形成三维结构[15]可以完成该工作。此类3D打印机已经被证明能够成功地设计、打印和测试医疗相关设备[16,17]。* Corresponding author. E-mail address: longzhicheng@pitt.edu (Z. Long), warrenr@pitt.edu (W.C. Ruder). Author name et al. / Engineering 2(2016) xxx–xxx197在这项研究中，我们选择了一种可以检测铜绿假单胞菌（Pseudomonas aeruginosa）的群体信号的稳定可靠方法，这是合成生物学中常用的方法[图1(a)]。活体生物传感器由工程化的大肠杆菌（Escherichia coli）组成，其被设计用来检测群体信号N-3-氧代十二烷酰-L-同型丝氨酸内酯（3O-C12）的存在。通过将铜绿假单胞菌的天然3O-C12信号通路的成分引入大肠杆菌，来构建活体生物传感器。在这里，转录因子LasR通过结合3O-C12和二聚体而充当信号转导子。然后，该复合物与同源启动子PLas [6,18,19]内的转录激活域结合，接着该转录激活域也被移植到大肠杆菌中。在我们的设计中，一旦结合，LasR就会促进红色荧光蛋白mCherry基因的表达。我们特别选择了3O-C12作为待检测的化学物质，因为它与医疗环境具有相关性[20–25]。有一个关于该方法临床相关性的例子，在农村和欠发达地区，可以在当地诊所获得有机体铜绿假单胞菌的肺部感染病例。在铜绿假单胞菌感染肺部后 [图1（b）]，可在肺痰标本中检出该菌。该痰液可收集并置于市售样品管中。因此，通过利用合成生物学工具，能够在现场立即分析这些样品的方法将是早期诊断的理想选择。在本文的解决方案中，合成生物传感器被置于一个可与普通样品管匹配的独特试管盖中[图1（b）]。这种独特的3D打印设备完全封装了活体生物传感器，允许其从实验室送出，同时还可以将传感器直接导入样本。当准备好分析痰液样本时，用手指向设备施加力，使细菌生物传感器从外壳中弹出并落入样品中。检测样品中由铜绿假单胞菌产生或已经存在于样品中的3O-C12，使活体生物传感器产生mCherry [图1（c）]并使样品变红，该颜色可通过荧光分析或肉眼来检测。因此，样本中的比色变化可以表明肺中是否存在铜绿假单胞菌。铜绿假单胞菌是囊性纤维化患者的一种条件致病菌，它会引起肺部和呼吸道的反复感染。目前的诊断技术通常采用酶联免疫吸附测定（enzyme-linked im-munosorbent assay，ELISA）[26]和实时聚合酶链反应polymerase chain reaction，PCR）测定[27]的形式，这两种检测方法要么价格昂贵，要么需要大量的培训才能进行。本文所描述的传感方法可以作为当前检测铜绿假单胞菌诊断技术简单且廉价的补充。由于细胞生物传感器可以生长在一个安全的、3D打印的封闭设备内，因此该设备可以用作在现场部署合成工程生物传感器的平台。图1. a）3O-C12通过使生物传感器能够持续表达一种可以与样品中的用GMO和3D打印试管盖对3O-C12进行现3O-C12场检测。分 子结合的LasR蛋白。一旦与3O-C12结合，LasR蛋白二聚化并与PLas启动子中的DNA区域相结合，从而促进mCherry的表达。（b）将生物传感器放置在3D3D打印装置的样品管中，然后按下设备盖，使生物传感器落入样品打印外壳中，提取生物样本并将其放置在连接到中。（c）生物传感器变红，表明样品暴露于3O-C12，然后可以使用流式细胞术量化该信号。直方图表示针对未被诱导的和3O-C12100 nmol·L?1样品（三个样品）的10 000个事件收集的数据。 2. 材料与方法2.1. 细胞培养与分子克隆本研究中使用的大肠杆菌菌株来源于大肠杆菌K12亲本菌株，见表1[28]。大肠杆菌细胞在37 ℃的溶菌肉汤（LB，赛默飞世尔科技公司，美国）培养基中分批培养[29]。使用100 μg·mL?1卡那霉素（赛默飞世尔科技公司，美国）作为选择抗性转化剂。为了进行克隆，使（（
一种可实现合成生物传感器现场部署的增材制造方法 - 图文
