合成生物学2-11-7课件

合成生物学(Syntheticbiology)

(概念、原理、应用)合成生物学应运而生…SyntheticBiologyWhatisSyntheticBiology?TakinganengineeringapproachtodesignandapplyingittoBiology使用工程策略设计并应用于生物学WhatisSyntheticBiology?1.Biology2.Chemistry3.Engineering4.Re-WritingBiologistsChemistsEngineers“Re-Writers”“Thecodeis3.6billionyearsold.It’stimeforare-write.”

-TomKnight合成生物学指人们将“基因”连接成网络，让细胞来完成设计人员设想的各种任务。例如把网络同简单的细胞相结合，可提高生物传感性，帮助检查人员确定地雷或生物武器的位置。再如向网络加入人体细胞，可以制成用于器官移植的完整器官。人工合成脊髓灰白质炎病毒cDNA美国纽约大学Wimmer实验室于2002年报道了化学合成脊髓灰白质炎病毒cDNA，并用RNA聚合酶将它转成有感染活力的病毒RNA。开辟了利用已知基因组序列，不需要天然模板，从化合物单体合成感染性病毒的先河。Venter实验室发展了合成基因组ΦX-174噬菌体基因是单链环状DNA，是历史上第一个被纯化的DNA分子，也是第一个被测序的DNA分子。ΦX-174噬菌体对动植物无害，是合适的合成研究对象。美国Venter实验室发展了合成基因组的工作，该实验室只用两周就合成了ΦX-174噬菌体基因(5,386bp)。Venter实验室的技术改进主要有：(1)用凝胶来提纯寡核苷酸以减少污染；(2)严格控制退火连接温度来防止与不正确的序列发生连接；(3)采用聚合酶循环装置来装配连结产物。合成生物学国际会议2004年6月在美国麻省理工学院举行了第一届合成生物学国际会议。会上除讨论了科学与技术问题外，还讨论了合成生物学当前与将来的生物学风险，有关伦理学问题，以及知识产权问题。随着这个领域的发展，对于合成生物学的安全性的考虑愈来愈多。现在不仅通过合成生成病毒，而且已经可以合成细菌。合成生物学开辟了设计生命的前景一方面有可能合成模仿生命物质特点的人工化学系统；另一方面也可能重新设计微生物如Keasling实验室向大肠杆菌中导入青蒿与酵母的基因，使大肠杆菌能在调节下合成青蒿素，从而显示了有效而价廉的治疗疟疾的前景合成生物学今后将能生成自然界不存在的新的微生物。应用示例Brenner提出向细胞DNA中掺入天然不存在的碱基来发展人工遗传系统,支持人工生命形式。合成生物学也将对生命起源，其他生命形式的研究作出贡献。控制生命目前，研究人员正在试图控制细胞的行为，研制不同的基因线路———即特别设计的、相互影响的基因。波士顿大学生物医学工程师科林斯已研制出一种“套环开关”，所选择的细胞功能可随意开关。加州大学生物学和物理学教授埃罗维茨等人研究出另外一种线路：当某种特殊蛋白质含量发生变化时，细胞能在发光状态和非发光状态之间转换，起到有机振荡器的作用，打开了利用生物分子进行计算的大门。维斯和加州理工学院化学工程师阿诺尔一起，采用“定向进化”的方法，精细调整研制线路，将基因网络插入细胞内，有选择性地促进细胞生长。维斯另一项大胆的计划是为成年干细胞编程促进某些干细胞分裂成骨细胞、肌肉细胞或软骨细胞等，让细胞去修补受损的心脏或生产出合成膝关节。尽管该工作尚处初级阶段，但却是生物学调控领域中重要的进展。J.CraigVenter：基因组替换成功利用基因组取代技术，将一种细菌改变为另一种与之亲缘关系较为紧密的另一细菌。这种由J.CraigVenter进行的“移植(transplantation)”技术，有望将合成基因组插入细胞，用于生产合成生命。用Mycoplasmamycoides的基因组取代与之关系密切的Mycoplasmacapricolum的基因组C.Lartigueetal."Genometransplantationinbacteria:Changingonespeciestoanother"Science,June28,2007.人类历史上第一个人造染色体合成成功美科学家称“人造生命”技术已被掌握最具争议的美国著名科学家克雷格·文特尔宣布，他的研究小组已经合成出人类历史上首个人造染色体，并有可能创造出首个永久性生命形式，以此作为应对疾病和全球变暖的潜在手段。该研究部分由美国能源部出资，希望藉此研制出新型环保燃料。由文特尔召集，诺贝尔医学奖获得者汉密尔顿·史密斯领导的研究小组在这方面已经进行了5年研究。文特尔已用化学药品在实验室中研制出一种合成染色体。“这是人类自然科学史上一次重大进步，显示人类正在从阅读基因密码走向有能力重新编写密码，这将赋予科学家新的能力，从事以前从未做过的研究。”他希望这项突破有助于发展新能源，应对气候变化造成的负面影响。如创造出具有特殊功能的新微生物，可被用作替代石油和煤炭的绿色燃料，或用来帮助清除危险化学物质或辐射等；还可用来合成能吸收过多二氧化碳的细菌，为解决气候变暖贡献力量。然而制造永久生命形式的前景极具争议性，有可能激起道德、伦理等方面的激烈辩论。加拿大生物伦理学组织ETC团体主任帕特·穆尼说，文特尔制造出了“一个基架，在此基架上人们几乎可以制造出任何东西”，“它可以用于研究新型药物，也可以用于对人类产生巨大威胁的生物武器”。《用化学合成的基因组构建一个细菌细胞》Venter的实验

实验对象：蕈状支原体。支原体是已知的可以自由生活的最小生物,也是最小的原核细胞。是一种原核微生物,内部结构很简单，基因组仅有一百多万碱基对，远小于真核生物基因组十亿级的碱基数量，这也是Venter选择操作它的原因。Venter早在1995年就对生殖支原体测序，并致力于研究维持自由生命的最小基因组。在2008年，Venter的团队合成了长达59万碱基对的生殖支原体基因组。此后，他们选择生长速度更快的蕈状支原体来做实验。如果仅仅从技术上来说，Venter做了一个无懈可击的实验，“人造生命”思路和流程都做得无懈可击。三个步骤：合成、组装和移植合成：蕈状支原体的基因组是一条大片段的DNA分子，序列是A、T、G、C四种脱氧核糖核苷酸的排列组合。通过实验确定维持其生命周期的最小基因组，并加上4个“水印基因”作为标记。用计算机精确计算需要合成DNA分子序列，并用化学方法合成A、T、G、C碱基，并使其按所要求序列延伸。这是它被称为“人造生命”或者“化学合成”的关键。Venter用化学方法合成了一千多个约1kb的DNA片段，作为这次组装的基本材料。组装：因为合成生物学技术上的局限，不能直接合成上万碱基对的DNA大分子，所以Venter等人巧妙地借助啤酒酵母和大肠杆菌的帮助，把1Kb的DNA分子有序准确的连成超过1000kb的片段。移植：

Venter等把这个合成基因组移植到不含限制性酶切系统的山羊支原体中，基因组能使用后者的酶系统进行自我复制，经过多代繁殖后，长成的菌落已经纯粹由蕈状支原体组成。Venter：“创造了一个计算机为父母的生命”JCVI：将8个由60个核苷酸组成的DNA片段，

首次人工合成实验老鼠的线粒体基因组使用8个只含有60个核苷酸的DNA片段，让它们同酶和化学试剂的混合物相结合，在50℃下孵化1小时，5天内合成出了实验鼠的线粒体基因组，得到的基因组能够纠正具有线粒体缺陷的细胞内的异常。

用途：生物能源、生物除污…Venter下一步的计划就是合成某种海藻基因组，这种新型海藻可以通过光合作用把空气中的二氧化碳转化成汽油或者柴油等清洁能源，从而有效解决目前的气候变化和能源危机。疫苗、药物、生物能源、生物除污等WhatisSyntheticBiology?——从原理角度来看SyntheticBiologyUndergraduatesinSyntheticBernationalGenetically

EngineeredMachinesLegoAssemblyforDNAPartsSelf-organizedPatternFormationWhatcanyoumakeinSB?ArsenicDetector脓毒症砷ModifyinglifeBiotechnology–Techniquesthatuselivingorganismsorpartsoforganismstoproduceavarietyofproducts(frommedicinestoindustrialenzymes)GeneticEngineering–Introductionofgeneticchanges(add,modify,delete)intoanorganismtoachievesomegoalSyntheticBiology–Createnovelbiologicalfunctionsandtoolsbymodifyingorintegratingwell-characterizedbiologicalcomponents(i.e.genes,promoters)intohigherordergeneticnetworksSyntheticBiologyHistory1970–Firstgenesynthesizedfromscratch(alaninetRNA)1978–NobelprizeawardedtoWernerArber,DanielNathansandHamiltonSmithforthediscoveryofrestrictionenzymes1978(BoyeratUCSF)–AsyntheticversionofthehumaninsulingenewasconstructedandinsertedintothebacteriumE.coli.1980–KaryMullisinventsPCR1991–Affymetrixchip-basedoligonucleotidesynthesis2003–FirstiGEMcompetition,creationofstandardizedpartslibrariesatMITBiotechnology1.0ResearchWorkflow1.Concept2.CollectDNAfragments(PCR,isolation,vendors,etc)6.Transform7.Test3.Benchwork5.VerifyDNA4.SequenceDNAsynthesiscostsaredroppingForexamplethebacteriaMycoplasmagenitaliumhasthesmallestgenomeoutofalllivingcells:517genesover580kb.Minimalcostsofoligocreation(notincludingerror-checking):Mid1990s:$1/bp=$580,000Circa2000:$0.35/bp=$203,0002006:$0.11/bp=$63,800Ambitiouspredictionofnot-too-distantfuture(Churchetal,2004):$0.00005/bp=$29SynthesislengthsareincreasingCommercialDNASynthesisCompaniesDataSource:RobCarlson,UofW,SeattleBioneerSouthKoreaCinnagenTehran,IranTakaraBiosciencesDalian,ChinaInqabaBiotecPretoria,SouthAfricaFermentasVilnius,LithuaniaBioS&T,AlphaDNA,BiocorpMontreal,CanadaGENEARTRegensberg,GermanyMWGBangalore,IndiaZelinskyInstituteMoscow,RussiaScinoPharmShan-hua,TaiwanGenosphereParis,FranceBiolegioMalden,NetherlandsAmbionAustin,TexasBiosearchNovato,CaliforniaBio-SynthesisLewisville,TexasChemgenesWilmington,Mass.BioSpringFrankfurtamMain,GermanyBiosourceCamarillo,CADharmaconLafaette,Co.CyberGeneABNovum,SwedenCortecDNAKingston,Ontario,CAEurogentecBelgium,U.K.DNATechnologyAarhus,DenmarkGenemedSynthesisS.SanFrancisco,CADNA2.0MenloPark,CAMetabionMunich,GermanyMicrosynthBalgach,SwitzerlandJapanBioServicesJapanBlueHeronBiotechnologyBothell,WAGeneworksAdelaide,AustraliaImperialBio-MedicChandigarh,IndiaBioserveBiotechnologiesHyderabad,IndiaGenelinkHawthorne,NY.DNASynthesis(Caruthersmethod)ErrorRate:1%0.9950=0.60300secondsperstepMicroarrayoligonucleotidesynthesisThepowerofparallelismChip-basedversuslinearsynthesisOligonucleotidessynthesizedSingle-strandedfragmentsof50-90nucleotides3’-overlappingnextfragmentby17nucleotides(Tmcalculated52-56°)Steps1to5involvemultipleroundsofPCR(heatingto95°,coolingto56°,andPCRat72°).Numberofroundsdependsonnumberoffragments.CarriedoutbyPCRmachine.Finalstepofamplificationofcompletegenedrivenbyuseofexcessofterminalsingle-strandedfragmentsPCR-basedoligoligationIntheory,thescaleofsynthesisisunlimitedBiotechnology2.0ResearchWorkflow1.Concept2.Design/debug/test4.Designoligos6.Transform7.Test5.SynthesizeDNA3.RuncodeWhataretheimplicationsofDNAsynthesiscapacity+freedomofinformation?Theproblem:“DualUse”ResearchDualuseresearchincludeslifesciencesresearch:WithlegitimatescientificpurposeThatmaybemisusedtoposeabiologicthreattopublichealthand/ornationalsecurity.Howeasyisittogetthistechnology?Whatcanwedo?NumberofIndividualsIndividual’sIntenthonorabledishonorableBinLadenGenetics,Inc.DisgruntledResearcherGarageBio-HackerBasicResearcherRiskspectrumBasiclogiccircuitsBorrowingfromelectricalengineeringProteinExpressionBasicsRNApolymerasebindstopromoterRNAPtranscribesgeneintomessengerRNARibosometranslatesmessengerRNAintoproteinZZPromoterZGeneProteinTranscriptionRNAPolymeraseDNATranslationMessengerRNARegulationThroughRepressionandInductionRepressorproteinscanbindtothepromoterandblocktheRNApolymerasefromperformingtranscriptionTheDNAsitenearthepromoterrecognizedbytherepressoriscalledanoperatorThetargetgenecancodeforanotherrepressionproteinenablingregulatorycascadesZPromoter&OperatorZGeneRGeneRRRPromoterTranscriptionTranslationDNABindingRNAPolymeraseLogicCircuitsProteinsarethewires/signalsPromoters+decayimplementthegatesAnyfinite-statedigitalcircuitcanbebuiltForexample,XorYZXYR1ZR1R1XYZ=genegenegeneTranscription-BasedInverterProteinconcentrationsareanalogoustoelectricalcurrentBUT…proteinsdonotfunctioninanisolatedsystemandneedtobeunique0110RRZSimpleInverterModelROperatorZGeneZRCooperativityCooperativeDNAbindingiswherethebindingofoneproteinincreasesthelikelihoodofasecondproteinbindingCooperativityaddsmorenon-linearitytothesystemIncreasesswitchingsensitivityImprovesrobustnesstonoiseZPromoter&OperatorZGeneRGeneRRRPromoterTranscriptionTranslationCooperativeDNABindingRNAPolymeraseRCooperativeInverterModelRROperatorZGeneZRBioCircuitComputer-AidedDesignSPICEBioSPICEsteadystatedynamicsintercellular

BioSPICE:aprototypebiocircuitCADtoolsimulatesproteinandchemicalconcentrationsintracellularcircuits,intercellularcommunicationsinglecells,smallcellaggregatesGeneticCircuitElementsinputmRNAribosomepromoteroutputmRNAribosomeoperatortranslationtranscriptionRNApRBSRBSABioSPICEInverterSimulationinputoutputrepressorpromoterTheyworkinvivo

Flip-flop(Gardner&Collins,2000)Ringoscillator(Elowitz&Leibler,2000)However,cellsareverycomplexenvironmentsCurrentmodelingtechniquespoorlypredictbehavior“ProofofConcept”Circuitstime(x100sec)[A][C][B]B_S_RA_[R][B]_[S][A]time(x100sec)time(x100sec)RS-Latch(“flip-flop”)RingoscillatorCellularLogicSummaryCurrentsystemsarelimitedtolessthanadozengatesThreeinverterringoscillator(Elowitz,2000)RSlatch(Gardner,2000)Inter-cellcommunication(Weiss,2001)Anaturalrepressor-basedlogictechnologypresentsseriousscalabilityissuesScavengingnaturalrepressorproteinsistimeconsumingMatchingnaturalrepressorproteinstoworktogetherisdifficultCellularLogicSummarySophisticatedsyntheticbiologicalsystemsrequireascalablecellularlogictechnologywithgoodcooperativityZinc-fingerproteinscanbeengineeredtocreatemanyuniqueproteinsrelativelyeasilyZinc-fingerproteinscanbefusedwithdimerizationdomainstoincreasecooperativityAcellularlogictechnologyofonlyzinc-fingerproteinsshouldhopefullybeeasiertocharacterizeSingleZinc-FingerStructureDNAThreeBaseRecognitionRegionZincAtomAlphaHelixTwoBetaSheetsPoly-FingerZFPsA.C.Jamieson,J.C.Miller,andC.O.Pabo.Drugdiscoverywithengineeredzinc-fingerproteins.NatureReviewsDrugDiscovery,May2003ComplexsystemsQ:Butifwedon’tfullyunderstandalltherulesofbiology,howcanwecreateanythingmorethanbasicsystems?A:Wecanpressourlimitsbymodularizingandsimplifyingasmuchaspossible.StandardizationofComponentsPredictableperformanceOff-the-shelfMechanicalEngineering(1800s)&themanufacturingrevolution(e.g.HenryFord)AbstractionInsulaterelevantcharacteristicsfromoverwhelmingdetailSimplecomponentsthatcanbeusedincombinationFromPhysicstoElectricalEngineering(1900s)DecouplingDesign&FabricationRulesinsulatingdesignprocessfromdetailsoffabricationEnableparts,device,andsystemdesignerstoworktogetherVLSIelectronics(1970s)EnablingSyntheticBiologyCharacterizationCataloginput-outputcharacteristicsofexistingandnewparts/devicesStandardizationPhysicalconnectionsFunctionalconnectionsPerformanceAPoPSINPoPSOUTSBworksviathreelayersofabstractionDevicesPartsSystemsAbstractioninbiologyDevicesPartsSystemsBarriers-Technological-Legal-EthicalSyntheticBiology:IntellectualPropertyRelationshipofsyntheticbiologytointellectualpropertylawhasbeenlargelyunexplored.Therelevantresearchspacealreadycontainsbroadpatentsonfoundationaltechnology.Syntheticbiologycommons?Toolsofopensource–propertyrightscoupledwithvirallicensingSyntheticBiology:IntellectualPropertyWhatispatentableand/orcopyrightable?BroadbiologicalfunctionsSpecificsequencesSpecificusesSourcesofuncertaintyinsyntheticbiologyasrelatedtoIPRdefinitionsWhatareeffectsofalternatedefinitionsofwhatispatentableandcopyrightableon:Developmentoffield?Efficiency?Justice?SyntheticBiology:IntellectualProperty

Patentsonfundamentalideasinsyntheticbiology

Example:Apatentontheideaofabiologicalpart:apieceofDNAwithspecificfunctionthatcanbecombinedwithanotherpartinapredefinedfashion.Suchapatentwouldbeimpossibletocircumvent.Itrepresentsafundamentalconceptthatunderpinssyntheticbiology.SeeStanfordpatentonSystemandmethodforsimulatingoperationofbiochemicalsystems.UnitedStatesPatent5914891

SyntheticBiology:IntellectualProperty

Patentsonfundamentalbiologicalfunctions

Example:Apatentonagenetically-encodedinverterSuchapatentwouldbealmostimpossibletocircumventbecauseitrepresentsabasicbiologicalfunctionthatisofuseinarangeofsyntheticbiologicalsystems.SeeUSDeptofHealthpatentonMolecularcomputingelements,gatesandflip-flops.UnitedStatesPatent6774222

SeeBostonUniversitypatentonMulti-stategeneticoscillator.UnitedStatesPatent6737269

SeeBostonUniversitypatentonBistablegenetictoggleswitch.UnitedStatesPatent6841376

SeeBostonUniversityparentonAdjustablethresholdswitch.UnitedStatesPatent6828140

SyntheticBiology:IntellectualProperty

Patentsonclassesofbiologicalmoleculeswithaparticularfunction

Example:ApatentontheuseofzincfingerproteinstobindaspecificsequenceofDNA.SuchapatentisnotimpossibletocircumventbecausethereareotherproteinsthatbindDNAandthatcouldbeengineeredtobindnewsequences.SeeMITpatentonPolyzincfingerproteinswithimprovedlinkers.UnitedStatesPatent6903185

SeeScrippsResearchInstitutepatentonZincfingerbindingdomainsforGNN.UnitedStatesPatent6610512

SeeSangamoBiosciences,Inc.patentonRegulationofendogenousgeneexpressionincellsusingzincfingerproteins.UnitedStatesPatent6607882

SyntheticBiology:IntellectualProperty

Patentonaparticularbiologicalmolecule.

Example:Apatentonthesequenceofaparticularproteinthatsenseslightandtransmitsasignalintothecell.Suchapatentwouldlikelybefairlyeasytocircumventbecausethereareprobablyafewaminoacidsthatcouldbechangedintheproteinsuchthatitwoulditwouldstillbefunctionalyetnothavetheexactsamesequenceasspecifiedinthepatent.Thereareexceptionstothisrule:Someproteinsthathavebeensooptimizedforaspecificfunctionthatanymutationinthesequencecanleadtolessfunctionality(e.g.,thepeptidedrugZiconitide).OpencommonsofbiologicalfunctionsOpen-accessbiology?Whenatechnologyisproprietary,boththeabilityandinterestinexamining&troubleshootingproblemsisrestrictedtothosewiththeIPMightopen-accessbiologygenerateahigherqualityproduct?Orwoulditstifleinnovationthroughalackofinterest?ProgrammedOrganisms(编程性物种)Super-efficientagricultureviaalterednutrientuptake(nitrogenfixingplants,etc)Controlledcropmaturing(countdays)ChemicallycontrolledpetsBiologicalrobotsBeneficialbacterialinfectionsprogrammedtoaugmentimmunity,provideneededvitamins,etc.CellsthatcirculateinthebodyasanextensionofimmunesystemSyntheticBiologyApplicationsSmartMaterials(聪明材料)Livingself-repairingmaterials(自我修复)NewdevicesandassemblytechnologiesNanofabricationofmicroandmacromaterialsEnergyproductionandstorage(能量产生与储存)NewbiologicalpathwaysSyntheticBiologyApplicationsMedicalMolecularmedicaldevicesReversalofaging(返老还童)Diseasefighting(抗病)Implantablelivingbatteryformedicaldeviceoutofelectriceelcells.Humansthatphotosynthesize(人类光合成)SyntheticBiologyApplicationsSensors(传感器)SmartsensorsUsecellstoread,process,outputinformationDetectarbitrarysubstancesSelf-reproducingchemical/radioactivitysensorsDetectbiotoxinsandencapsulate.flashwhenitdoes.Responsivematerials(e.g.,oillubricantsbydesign/need)ToolstomeasureconcentrationofproteinincellEcosystemdebugger(read/write)IntelligentBiosensors(智能型传感器)SyntheticBiologyApplicationsTerraformingCreatinglifeonotherworlds仿地成形（尤指在科幻小说中，在外星球创建仿地球的生存环境，以使人类能够生存）NewlydiscoveredarchaeaExtremophiles:

ThermophilestoPsychrophilesLifeasahyperthermophile

(hightemperature)Problem:AthighT,membranesbecometoofluidandpermeable.Adaptation:ChangethelipidstobemorewaxyProblem:atT>70C,DNA&RNAstartstodegradeAdaptation:Increasethesaltsolutionwithinthecelltoprotectthem.Adaptation:GenomicbiastowardsthemorestableG-CbasepairsProblem:Proteinsdon’tfoldaswellathighTAdaptation:Evolvemorestably-foldingproteins(e.g.,tighterhydrophobiccores)Lifeasapsychrophile(lowtemperature)Problem:AtlowT,membranesbecometoostiff.Adaptation:Changethelipidstobemoregreasy.Problem:Waterfreezes,andicecrystalsbreakcellsAdaptation:Use“antifreeze”moleculestoinhibitcrystalgrowthProblem:NotenoughenergytoovercomechemicalbarriersAdaptation:EvolvemoreactiveenzymesLifeasanextremophileOxyphiles–organismsthatloveoxygen(需氧)Problem:Oxygenreactionsproducereactivespecieslikeoxygenfreeradicals,Adaptation:Developanti-oxidants(e.g.,somevitaminsandflavinoids)Halophiles–organismsthatliveinhigh-saltenvironments(高盐)Problem:ReverseosmoticpressuredesiccatescellsAdaptation:Producesomethinginsidecell(usu.glycine,sometimespotassium)whoseosmoticpressurebalancesthatofsaltoutsidecell.Acidophiles/Alkalophiles–organismsthatloveacidic/basicconditions(酸碱)Problem:ProteinscanbedegradedbychangesinpH(e.g.,ceviche)Adaptation:UsemolecularpumpstokeeptheinteriorpHclosetoneutral.Xerophiles–organismsthatliveinextremelydryenvironments(干燥)Problem:waterevaporates.Adaptation:Protectsurface(desertvarnish)Adaptation:Increaseinteriorosmoticpressure,orletcelldryout…Problem:Oxygenfreeradicalsaccumulateascelldries;DNAbreaksAdaptation:Fixit!Sidebenefit:extremeradiationresistance[D.Radiodurans:incredibleresistance]OtherextremophilesDesertVarnish–existsinthedriestplacesonEarthVarnishincludesbacteriathat:Arrangeclayandmanganeseabovethemtoshieldthemfromtheelements;oxidizeMntoproduceATPAregreatforshowingwherepollutantsinwaterexistorwhereoff-roadvehiclesstirupalkalinedust.Lichens–asymbiosisoffungiandalgaeDryoutcompletelyandphotosynthesizeonlywhenwetThefirststepincreatingsoiloutofrock(e.g.,SierraNevada:polishedbyglaciers12kyrago,heavilywoodednow.)Edible!(Manna?)XerophilesPiezophiles–organismsthatliveathighpressure(高气压)Pressureincreasesby1atm(=15poundspersquareinch)every10metersinwater,orevery5metersinrock.Benefit:Waterisliquidforahigherrangeoftemperaturesasthepressuregoesup…thisallowsliquidwatertotensofkilometersdepth[Tgoesup25Cperkmincrust…so121C=about4km]Problem:PressurechangesthepackingofDNAandmembranelipidsProblem:Pressureinhibitsreactionsthatlowerthedensity(moreproductsthanreactants)Adaptation:?LifeinVacuum1964:Surveyor3camerainspacefor2.6years,unprotected.OnreturningfromtheMoon,viablestreptococcusbacteriaareculturedfromit!MoreextremophilesLongevityViablemicrobesfromicecores(LakeVostok)–upto20MyrFrombeeabdomensinamber–25MyrFromsaltinsaltmines–manyMyr(controversial)Multicellularextremophiles?

Tartigrades(waterbears):inadry(tun)state,canwithstandtemperaturesupto151C,X-rays,vacuum,andpressuresof6000atmospheres.Lifewithoutlight?Autolithotrophiccommunities:(SLiMe)Basaltrock&water:hasC,N,O,H,S–justneedenergyEnergyfromoxidationofS&HandreductionofSandnitrates.Note:lifehadtobelikethisbeforephotosynthesiswasinvented.MoreamazinglifeSummaryCreatingbiologicalcircuitsmayteachusasmuchaboutlifeastryingtoreverse-engineerthem(learnbydoing)ThekeystoSBareabstraction,isolationofdesign&fabricationprinciplesandmodularitySyntheticBiologySergioPeisajovichLimLabJune2007SyntheticBiologyWhatisSyntheticBiology?Itisanemergingfieldofbiologythataimsatdesigningandbuildingnovelbiologicalsystems.Thefinalgoalistobeabletodesignbiologicalsystemsinthesamewayengineersdesignelectronicormechanicalsystems.Whydoweneedit?“WhatIcannotcreate,Idonotunderstand.”

-RichardFeynman无法创造的东西，我无法理解——只有通过创造才能理解。不能理解的东西，我无法创造。WhatIcannotcreateIdonotunderstand.——美国物理学家理查德·费曼SyntheticBiologyWhydoweneedit?CellsaretheultimateChemicalFactory.SyntheticBiology1-BiologyishierarchicalIsitachievable?SyntheticBiology2-BiologyisModularIsitachievable?SyntheticBiologyHierarchyandModular(recurrent)organizationallowsbiologytobeunderstandableandsyntheticbiologytobepossible.Isitachievable?SyntheticBiologyApossiblehierarchyforsyntheticbiologySyntheticBiologyBiologicalComponents:1-PartsSyntheticBiologyBiologicalComponents:2-DevicesSyntheticBiologyBiologicalComponents:3-SystemsorModulesSyntheticBiologyBiologicalComponents:3-SystemsorModulesBasuetal(2005)Nature,434:1130-4SyntheticBiologyBiologicalComponents:3-SystemsorModulesSyntheticBiologyBiologicalComponents:3-SystemsorModulesSyntheticBiologyForsyntheticbiologytobecomeaformofengineeringitwillbenecessarytoachieveprecisionandreliability.Factorspreventingthis: 1-Incompleteknowledgeof biology. 2-Inherentfunctionaloverlap (partswithmany-someunknown- functions,someofwhichare detrimentaltothegoalinmind. 3-Incompatibilitybetweenparts. 4-Partsfunctionalitydependson context.SyntheticBiologyasEngineering2-CIrepressesexpressionofunrelatedhostgenes3-LuxRinteractswithCIandblocksitsfunction4-GFPisnon-fluorescentinhostSyntheticBiologySyntheticBiologyasEngineeringStandardPartsPartsshouldnothavemultiplefunctions(OnesubunitofT7phageDNApolymeraseisactuallyE.colithioredoxin)PartsshouldnotencodemultiplefunctionsSyntheticBiologySyntheticBiologyasEngineeringStandardPartsDifferentpartsshouldbecompatiblePartsshouldworkindifferentcontextsSyntheticBiologySyntheticBiologyasEngineeringStandardPartsStandardizedpartscouldbeeasilyexchangedbetweendifferentdevices(aswellasbetweendifferentlaboratories)SyntheticBiologySyntheticBiologyasEngineeringAbstractionDNATGCATGCTGATATACGGCTCGATPartsDevicesSystemsYeast&CloningSergioPeisajovichLimLabJune2007ExperimentalLabWhyYeast?TheyeastSaccharomycescerevisiae(alsocalled“baker’syeast”)isprobablytheidealeukaryoticmicroorganismforbiologicalstudies.Yeastgenome:fullysequencedandeasytomanipulate.Basicmechanismsofyeastcellbiology(suchasDNAreplication,recombination,celldivisionandmetabolism)

arehighlysimilartothatofhigherorganisms(includinghumans).ExperimentalLabYeastLifeCycleExperimentalLabYeast:IdealPlatformforSyntheticBiologyAddparts,devicesorevenmodules(inan“extra-genomic”format-plasmid-based-or“integrating”themwithintheyeastgenome.Deletespecificyeastgenes,toremove“background”orinterference.Add“reportergenes”tomonitorinrealtimethefunctionofthesyntheticparts/devices/modulesunderstudy.Lifecyclefastenoughsothatwecoulddoallthesegeneticmanipulationsinareasonableamountoftime.Parts/Devices/ModulesarebuiltinbacteriaEmptyinitialplasmidPlasmidcodingthedesireddeviceTransformintoYeastExperimentalLabYeast:Addingparts…inplasmidsExperimentalLabYeast:Addingparts…inplasmidsgrowthinselectivemediumExperimentalLabYeast:Addingparts…intothegenomeHomologousrecombinationallowsgenomicintegration,butwestillneedtoselect:ExperimentalLabYeast:Addingparts…intothegenomePart/Device/ModuleURA3plasmidDigestwithspecificrestrictionenzymePart/Device/ModuleplasmidLinearDNA,readyforyeasttransformationandintegrationPart/Device/ModuleURA3*HomologousRecombinationYeastChromosomeIncomingLinearDNAURA3*URA3Part/Device/ModuleIntegration(Notethat2copies,onedefectiveandonefunctional,ofthemarkeraregenerated)YeastChromosomeExperimentalLabYeast:Addingparts…intothegenomeURA3plasmidURA3PCRproductLinearDNA,readyforyeasttransformationandintegrationyfgHomologousRecombinationYeastChromosomeURA3Integration(yfgisnowdisrupted)YeastChromosomeURA3Part1plasmidplasmidPart1plasmidplasmidPart2Part1plasmidplasmidPart2Part3ExperimentalLabCombinatorialCloningABBCCDADADExperimentalLabCombinatorialCloningBasedonTypeIIsrestrictionenzymesABBCCDADADADCombinatorialCloningExperimentalLabCombinatorialLibrariesExperimentalLabSyntheticBiologyasEngineeringEngineeringNegativeFeedbackLoopsNegativeEffectorstobeused:OspF(MAPKPhosphothreonineLyase)YopJ(MAPKKSer/Thracetylase)YopH(MAPKTyrphosphatase)Promoterstobeused:Constitutiveexpression(Adhp,CycIp,Ste5p)Induciblebypathwayactivation(STLp,Fig1p)Protein-interactiondomains:LeucineZippers(highandmediumaffinities,somewithdegradationmotif)PromTagEffectorZipperTermExperimentalLabSyntheticBiologyasEngineeringEngineeringNegativeFeedbackLoops1-CombinatorialCloninginBacteria2-TransferConstructsintoYeast3-AnalyzePathwayBehaviorExperimentalLabSynthetic