合成生物學

Syntheticbiology

(概念、原理、應用)張成崗軍事醫學科學院放射與輻射醫學研究所2011.11.02人工染色體(技術)BAC(細菌人工染色體)：Bacteria…以細菌作為對象，將DNA片段與質粒重組后轉入細菌中繁殖YAC(酵母人工染色體)：Yeast…以酵母作為對象PAC(噬菌體人工染色體)：Phagemid…以噬菌體作為對象TAC(可轉化的細菌人工染色體)MAC(哺乳類人工染色體)…合成生物學應運而生…SyntheticBiologyWhatisSyntheticBiology?TakinganengineeringapproachtodesignandapplyingittoBiology使用工程策略設計并應用于生物學WhatisSyntheticBiology?1.Biology2.Chemistry3.Engineering4.Re-WritingBiologistsChemistsEngineers“Re-Writers”“Thecodeis3.6billionyearsold.It’stimeforare-write.”

-TomKnightBiology“Testmodelsbybuildingthem”合成生物學指人們將“基因”連接成網絡，讓細胞來完成設計人員設想的各種任務。例如把網絡同簡單的細胞相結合，可提高生物傳感性，幫助檢查人員確定地雷或生物武器的位置。再如向網絡加入人體細胞，可以制成用于器官移植的完整器官。人工合成脊髓灰白質炎病毒cDNA美國紐約大學Wimmer實驗室于2002年報道了化學合成脊髓灰白質炎病毒cDNA，并用RNA聚合酶將它轉成有感染活力的病毒RNA。開辟了利用已知基因組序列，不需要天然模板，從化合物單體合成感染性病毒的先河。Wimmer從裝配平均長度為69bp的寡核苷酸入手，結合了化學合成與無細胞體系的從頭合成，用了3年時間完成了這個劃時代的工作。Venter實驗室發展了合成基因組ΦX-174噬菌體基因是單鏈環狀DNA，是歷史上第一個被純化的DNA分子，也是第一個被測序的DNA分子。ΦX-174噬菌體對動植物無害，是合適的合成研究對象。美國Venter實驗室發展了合成基因組的工作，該實驗室只用兩周就合成了ΦX-174噬菌體基因(5,386bp)。Venter實驗室的技術改進主要有：(1)用凝膠來提純寡核苷酸以減少污染；(2)嚴格控制退火連接溫度來防止與不正確的序列發生連接；(3)采用聚合酶循環裝置來裝配連結產物。合成生物學國際會議2004年6月在美國麻省理工學院舉行了第一屆合成生物學國際會議。會上除討論了科學與技術問題外，還討論了合成生物學當前與將來的生物學風險，有關倫理學問題，以及知識產權問題。隨著這個領域的發展，對于合成生物學的安全性的考慮愈來愈多。現在不僅通過合成生成病毒，而且已經可以合成細菌。合成生物學開辟了設計生命的前景一方面有可能合成模仿生命物質特點的人工化學系統；另一方面也可能重新設計微生物如Keasling實驗室向大腸桿菌中導入青蒿與酵母的基因，使大腸桿菌能在調節下合成青蒿素，從而顯示了有效而價廉的治療瘧疾的前景合成生物學今后將能生成自然界不存在的新的微生物。應用示例Schultz實驗室研究向大腸桿菌蛋白質生物合成裝置中添入新組份，使之能通過基因生成非天然的氨基酸，結果取得了成功。但是要在真核細胞做到這一點還有難度。2003年，Schultz實驗室報道了一種向酵母加入非天然氨基酸密碼子的方法，成功地向蛋白質中導入了5種氨基酸。目前，能摻入到蛋白質的非天然氨基酸已有80多種。今后將可以直接向蛋白質導入順磁標記、金屬結合、光敏異構化等的氨基酸，促進蛋白質結構與功能的研究。應用示例Brenner提出向細胞DNA中摻入天然不存在的堿基來發展人工遺傳系統,支持人工生命形式。合成生物學也將對生命起源，其他生命形式的研究作出貢獻。控制生命目前，研究人員正在試圖控制細胞的行為，研制不同的基因線路———即特別設計的、相互影響的基因。波士頓大學生物醫學工程師科林斯已研制出一種“套環開關”，所選擇的細胞功能可隨意開關。加州大學生物學和物理學教授埃羅維茨等人研究出另外一種線路：當某種特殊蛋白質含量發生變化時，細胞能在發光狀態和非發光狀態之間轉換，起到有機振蕩器的作用，打開了利用生物分子進行計算的大門。維斯和加州理工學院化學工程師阿諾爾一起，采用“定向進化”的方法，精細調整研制線路，將基因網絡插入細胞內，有選擇性地促進細胞生長。發展方向維斯目前正在研究另外一群稱為“規則系統”的基因，他希望細菌能估計刺激物的距離，并根據距離的改變做出反應。該項研究可用來探測地雷位置(TNT：生物傳感器)。維斯另一項大膽的計劃是為成年干細胞編程促進某些干細胞分裂成骨細胞、肌肉細胞或軟骨細胞等，讓細胞去修補受損的心臟或生產出合成膝關節。盡管該工作尚處初級階段，但卻是生物學調控領域中重要的進展。J.CraigVenter：基因組替換成功利用基因組取代技術，將一種細菌改變為另一種與之親緣關系較為緊密的另一細菌。這種由J.CraigVenter進行的“移植(transplantation)”技術，有望將合成基因組插入細胞，用于生產合成生命。用Mycoplasmamycoides的基因組取代與之關系密切的Mycoplasmacapricolum的基因組C.Lartigueetal."Genometransplantationinbacteria:Changingonespeciestoanother"Science,June28,2007.人類歷史上第一個人造染色體合成成功美科學家稱“人造生命”技術已被掌握最具爭議的美國著名科學家克雷格·文特爾宣布，他的研究小組已經合成出人類歷史上首個人造染色體，并有可能創造出首個永久性生命形式，以此作為應對疾病和全球變暖的潛在手段。該研究部分由美國能源部出資，希望藉此研制出新型環保燃料。由文特爾召集，諾貝爾醫學獎獲得者漢密爾頓·史密斯領導的研究小組在這方面已經進行了5年研究。文特爾已用化學藥品在實驗室中研制出一種合成染色體。文特爾研究小組研制出的這種新型染色體即實驗室合成支原體(Mycoplasmalaboratorium)，是一種經過簡化拼接的生殖支原體(Mycoplasmagenitalium)DNA序列，他們將這種合成支原體移植到活細胞中，使之在細胞中起主控作用，變換成一種新的染色體。按照實驗計劃，最終這個染色體將控制這個細胞并變成一個新的生命形式。這種新單細胞生物體被命名為“合成器”，受381個基因控制，包含56萬個堿基對。這些基因是維持細菌生命所必備的，使它能夠攝食和繁殖。由于新的生物體是在現存生物體上搭建，其繁殖和新陳代謝仍然依賴原來生物體的胞內機制。從這一角度看，它并非完全意義上的新型生命形式。但這種給特定基因賦予特定任務的觀點已被眾多生物學家廣泛接受。“這是人類自然科學史上一次重大進步，顯示人類正在從閱讀基因密碼走向有能力重新編寫密碼，這將賦予科學家新的能力，從事以前從未做過的研究。”他希望這項突破有助于發展新能源，應對氣候變化造成的負面影響。如創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綠色燃料，或用來幫助清除危險化學物質或輻射等；還可用來合成能吸收過多二氧化碳的細菌，為解決氣候變暖貢獻力量。然而制造永久生命形式的前景極具爭議性，有可能激起道德、倫理等方面的激烈辯論。加拿大生物倫理學組織ETC團體主任帕特·穆尼說，文特爾制造出了“一個基架，在此基架上人們幾乎可以制造出任何東西”，“它可以用于研究新型藥物，也可以用于對人類產生巨大威脅的生物武器”。2009：Venter：Science把蕈狀支原體的基因組加以改造，使它能夠終移植到山羊支原體內，形成了一個新的蕈狀支原體細胞。這也是今年這篇科研論文的雛形，在國外的科學媒體上曾經引發熱烈的討論。2010年的重要大事：

“人造生命”誕生JohnCraigVenter攪亂了(生命)科學界《用化學合成的基因組構建一個細菌細胞》實驗對象：蕈狀支原體。支原體是已知的可以自由生活的最小生物,也是最小的原核細胞。是一種原核微生物,內部結構很簡單，基因組僅有一百多萬堿基對，遠小于真核生物基因組十億級的堿基數量，這也是Venter選擇操作它的原因。Venter早在1995年就對生殖支原體測序，并致力于研究維持自由生命的最小基因組。在2008年，Venter的團隊合成了長達59萬堿基對的生殖支原體基因組。此后，他們選擇生長速度更快的蕈狀支原體來做實驗。如果僅僅從技術上來說，Venter做了一個無懈可擊的實驗，“人造生命”思路和流程都做得無懈可擊。三個步驟：合成、組裝和移植合成：蕈狀支原體的基因組是一條大片段的DNA分子，序列是A、T、G、C四種脫氧核糖核苷酸的排列組合。通過實驗確定維持其生命周期的最小基因組，并加上4個“水印基因”作為標記。用計算機精確計算需要合成DNA分子序列，并用化學方法合成A、T、G、C堿基，并使其按所要求序列延伸。這是它被稱為“人造生命”或者“化學合成”的關鍵。Venter用化學方法合成了一千多個約1kb的DNA片段，作為這次組裝的基本材料。組裝：因為合成生物學技術上的局限，不能直接合成上萬堿基對的DNA大分子，所以Venter等人巧妙地借助啤酒酵母和大腸桿菌的幫助，把1Kb的DNA分子有序準確的連成超過1000kb的片段。移植：

Venter等把這個合成基因組移植到不含限制性酶切系統的山羊支原體中，基因組能使用后者的酶系統進行自我復制，經過多代繁殖后，長成的菌落已經純粹由蕈狀支原體組成。Venter：“創造了一個計算機為父母的生命”JCVI：將8個由60個核苷酸組成的DNA片段，

首次人工合成實驗老鼠的線粒體基因組使用8個只含有60個核苷酸的DNA片段，讓它們同酶和化學試劑的混合物相結合，在50℃下孵化1小時，5天內合成出了實驗鼠的線粒體基因組，得到的基因組能夠糾正具有線粒體缺陷的細胞內的異常。

用途：生物能源、生物除污…Venter下一步的計劃就是合成某種海藻基因組，這種新型海藻可以通過光合作用把空氣中的二氧化碳轉化成汽油或者柴油等清潔能源，從而有效解決目前的氣候變化和能源危機。疫苗、藥物、生物能源、生物除污等WhatisSyntheticBiology?——從原理角度來看SyntheticBiologyUndergraduatesinSyntheticBio.internationalGenetically

EngineeredMachines/registry/index.php/Main_PageLegoAssemblyforDNAParts/registry/index.php/Assembly:Standard_assemblySelf-organizedPatternFormationWhatcanyoumakeinSB?ArsenicDetector膿毒癥砷LectureoverviewWhatwe’vetalkedaboutsofarThestudyofbiologicalsystems,fromcomponentsandinteractionstodynamicsOverviewOurunderstandingofsystemsiscompletewhenwecandesignourownDNAsynthesistechnologiesModifyinglifeBiotechnology–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(編程性物種)Super-efficientagricultureviaalterednutrientuptake(nitrogenfixingplants,etc)Controlledcropmaturing(countdays)ChemicallycontrolledpetsBiologicalrobotsBeneficialbacterialinfectionsprogrammedtoaugmentimmunity,provideneededvitamins,etc.CellsthatcirculateinthebodyasanextensionofimmunesystemSyntheticBiologyApplicationsSmartMaterials(聰明材料)Livingself-repairingmaterials(自我修復)NewdevicesandassemblytechnologiesNanofabricationofmicroandmacromaterialsEnergyproductionandstorage(能量產生與儲存)NewbiologicalpathwaysSyntheticBiologyApplicationsMedicalMolecularmedicaldevicesReversalofaging(返老還童)Diseasefighting(抗病)Implantablelivingbatteryformedicaldeviceoutofelectriceelcells.Humansthatphotosynthesize(人類光合成)SyntheticBiologyApplicationsSensors(傳感器)SmartsensorsUsecellstoread,process,outputinformationDetectarbitrarysubstancesSelf-reproducingchemical/radioactivitysensorsDetectbiotoxinsandencapsulate.flashwhenitdoes.Responsivematerials(e.g.,oillubricantsbydesign/need)ToolstomeasureconcentrationofproteinincellEcosystemdebugger(read/write)IntelligentBiosensors(智能型傳感器)SyntheticBiologyApplicationsTerraformingCreatinglifeonotherworlds仿地成形（尤指在科幻小說中，在外星球創建仿地球的生存環境，以使人類能夠生存）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(高鹽)

Problem:ReverseosmoticpressuredesiccatescellsAdaptation:Producesomethinginsidecell(usu.glycine,sometimespotassium)whoseosmoticpressurebalancesthatofsaltoutsidecell.Acidophiles/Alkalophiles–organismsthatloveacidic/basicconditions(酸堿)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(高氣壓)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無法創造的東西，我無法理解——只有通過創造才能理解。不能理解的東西，我無法創造。WhatIcannotcreateIdonotunderstand.——美國物理學家理查德·費曼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