XXXX1102 周三下午 合成生物學(xué) NXP

合成生物學(xué)

Syntheticbiology

(概念、原理、應(yīng)用)張成崗軍事醫(yī)學(xué)科學(xué)院放射與輻射醫(yī)學(xué)研究所2011.11.02人工染色體(技術(shù))BAC(細(xì)菌人工染色體)：Bacteria…以細(xì)菌作為對象，將DNA片段與質(zhì)粒重組后轉(zhuǎn)入細(xì)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(zhuǎn)化的細(xì)菌人工染色體)MAC(哺乳類人工染色體)…合成生物學(xué)應(yīng)運(yùn)而生…SyntheticBiologyWhatisSyntheticBiology?TakinganengineeringapproachtodesignandapplyingittoBiology使用工程策略設(shè)計(jì)并應(yīng)用于生物學(xué)WhatisSyntheticBiology?1.Biology2.Chemistry3.Engineering4.Re-WritingBiologistsChemistsEngineers“Re-Writers”“Thecodeis3.6billionyearsold.It’stimeforare-write.”

-TomKnightBiology“Testmodelsbybuildingthem”合成生物學(xué)指人們將“基因”連接成網(wǎng)絡(luò)，讓細(xì)胞來完成設(shè)計(jì)人員設(shè)想的各種任務(wù)。例如把網(wǎng)絡(luò)同簡單的細(xì)胞相結(jié)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(wǎng)絡(luò)加入人體細(xì)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(zhì)炎病毒cDNA美國紐約大學(xué)Wimmer實(shí)驗(yàn)室于2002年報(bào)道了化學(xué)合成脊髓灰白質(zhì)炎病毒cDNA，并用RNA聚合酶將它轉(zhuǎn)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(jié)合了化學(xué)合成與無細(xì)胞體系的從頭合成，用了3年時(shí)間完成了這個(gè)劃時(shí)代的工作。Venter實(shí)驗(yàn)室發(fā)展了合成基因組ΦX-174噬菌體基因是單鏈環(huán)狀DNA，是歷史上第一個(gè)被純化的DNA分子，也是第一個(gè)被測序的DNA分子。ΦX-174噬菌體對動(dòng)植物無害，是合適的合成研究對象。美國Venter實(shí)驗(yàn)室發(fā)展了合成基因組的工作，該實(shí)驗(yàn)室只用兩周就合成了ΦX-174噬菌體基因(5,386bp)。Venter實(shí)驗(yàn)室的技術(shù)改進(jìn)主要有：(1)用凝膠來提純寡核苷酸以減少污染；(2)嚴(yán)格控制退火連接溫度來防止與不正確的序列發(fā)生連接；(3)采用聚合酶循環(huán)裝置來裝配連結(jié)產(chǎn)物。合成生物學(xué)國際會(huì)議2004年6月在美國麻省理工學(xué)院舉行了第一屆合成生物學(xué)國際會(huì)議。會(huì)上除討論了科學(xué)與技術(shù)問題外，還討論了合成生物學(xué)當(dāng)前與將來的生物學(xué)風(fēng)險(xiǎn)，有關(guān)倫理學(xué)問題，以及知識(shí)產(chǎn)權(quán)問題。隨著這個(gè)領(lǐng)域的發(fā)展，對于合成生物學(xué)的安全性的考慮愈來愈多。現(xiàn)在不僅通過合成生成病毒，而且已經(jīng)可以合成細(xì)菌。合成生物學(xué)開辟了設(shè)計(jì)生命的前景一方面有可能合成模仿生命物質(zhì)特點(diǎn)的人工化學(xué)系統(tǒng)；另一方面也可能重新設(shè)計(jì)微生物如Keasling實(shí)驗(yàn)室向大腸桿菌中導(dǎo)入青蒿與酵母的基因，使大腸桿菌能在調(diào)節(jié)下合成青蒿素，從而顯示了有效而價(jià)廉的治療瘧疾的前景合成生物學(xué)今后將能生成自然界不存在的新的微生物。應(yīng)用示例Schultz實(shí)驗(yàn)室研究向大腸桿菌蛋白質(zhì)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(jié)果取得了成功。但是要在真核細(xì)胞做到這一點(diǎn)還有難度。2003年，Schultz實(shí)驗(yàn)室報(bào)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(zhì)中導(dǎo)入了5種氨基酸。目前，能摻入到蛋白質(zhì)的非天然氨基酸已有80多種。今后將可以直接向蛋白質(zhì)導(dǎo)入順磁標(biāo)記、金屬結(jié)合、光敏異構(gòu)化等的氨基酸，促進(jìn)蛋白質(zhì)結(jié)構(gòu)與功能的研究。應(yīng)用示例Brenner提出向細(xì)胞DNA中摻入天然不存在的堿基來發(fā)展人工遺傳系統(tǒng),支持人工生命形式。合成生物學(xué)也將對生命起源，其他生命形式的研究作出貢獻(xiàn)?？刂粕壳?，研究人員正在試圖控制細(xì)胞的行為，研制不同的基因線路———即特別設(shè)計(jì)的、相互影響的基因。波士頓大學(xué)生物醫(yī)學(xué)工程師科林斯已研制出一種“套環(huán)開關(guān)”，所選擇的細(xì)胞功能可隨意開關(guān)。加州大學(xué)生物學(xué)和物理學(xué)教授埃羅維茨等人研究出另外一種線路：當(dāng)某種特殊蛋白質(zhì)含量發(fā)生變化時(shí)，細(xì)胞能在發(fā)光狀態(tài)和非發(fā)光狀態(tài)之間轉(zhuǎn)換，起到有機(jī)振蕩器的作用，打開了利用生物分子進(jìn)行計(jì)算的大門。維斯和加州理工學(xué)院化學(xué)工程師阿諾爾一起，采用“定向進(jìn)化”的方法，精細(xì)調(diào)整研制線路，將基因網(wǎng)絡(luò)插入細(xì)胞內(nèi)，有選擇性地促進(jìn)細(xì)胞生長。發(fā)展方向維斯目前正在研究另外一群稱為“規(guī)則系統(tǒng)”的基因，他希望細(xì)菌能估計(jì)刺激物的距離，并根據(jù)距離的改變做出反應(yīng)。該項(xiàng)研究可用來探測地雷位置(TNT：生物傳感器)。維斯另一項(xiàng)大膽的計(jì)劃是為成年干細(xì)胞編程促進(jìn)某些干細(xì)胞分裂成骨細(xì)胞、肌肉細(xì)胞或軟骨細(xì)胞等，讓細(xì)胞去修補(bǔ)受損的心臟或生產(chǎn)出合成膝關(guān)節(jié)。盡管該工作尚處初級(jí)階段，但卻是生物學(xué)調(diào)控領(lǐng)域中重要的進(jìn)展。J.CraigVenter：基因組替換成功利用基因組取代技術(shù)，將一種細(xì)菌改變?yōu)榱硪环N與之親緣關(guān)系較為緊密的另一細(xì)菌。這種由J.CraigVenter進(jìn)行的“移植(transplantation)”技術(shù)，有望將合成基因組插入細(xì)胞，用于生產(chǎn)合成生命。用Mycoplasmamycoides的基因組取代與之關(guān)系密切的Mycoplasmacapricolum的基因組C.Lartigueetal."Genometransplantationinbacteria:Changingonespeciestoanother"Science,June28,2007.人類歷史上第一個(gè)人造染色體合成成功美科學(xué)家稱“人造生命”技術(shù)已被掌握最具爭議的美國著名科學(xué)家克雷格·文特爾宣布，他的研究小組已經(jīng)合成出人類歷史上首個(gè)人造染色體，并有可能創(chuàng)造出首個(gè)永久性生命形式，以此作為應(yīng)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(huán)保燃料。由文特爾召集，諾貝爾醫(yī)學(xué)獎(jiǎng)獲得者漢密爾頓·史密斯領(lǐng)導(dǎo)的研究小組在這方面已經(jīng)進(jìn)行了5年研究。文特爾已用化學(xué)藥品在實(shí)驗(yàn)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(shí)驗(yàn)室合成支原體(Mycoplasmalaboratorium)，是一種經(jīng)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(xì)胞中，使之在細(xì)胞中起主控作用，變換成一種新的染色體。按照實(shí)驗(yàn)計(jì)劃，最終這個(gè)染色體將控制這個(gè)細(xì)胞并變成一個(gè)新的生命形式。這種新單細(xì)胞生物體被命名為“合成器”，受381個(gè)基因控制，包含56萬個(gè)堿基對。這些基因是維持細(xì)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(xiàn)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(nèi)機(jī)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(wù)的觀點(diǎn)已被眾多生物學(xué)家廣泛接受?！斑@是人類自然科學(xué)史上一次重大進(jìn)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(xué)家新的能力，從事以前從未做過的研究。”他希望這項(xiàng)突破有助于發(fā)展新能源，應(yīng)對氣候變化造成的負(fù)面影響。如創(chuàng)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(xiǎn)化學(xué)物質(zhì)或輻射等；還可用來合成能吸收過多二氧化碳的細(xì)菌，為解決氣候變暖貢獻(xiàn)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(xué)組織ETC團(tuán)體主任帕特·穆尼說，文特爾制造出了“一個(gè)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(chǎn)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(nèi)，形成了一個(gè)新的蕈狀支原體細(xì)胞。這也是今年這篇科研論文的雛形，在國外的科學(xué)媒體上曾經(jīng)引發(fā)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(xué)界《用化學(xué)合成的基因組構(gòu)建一個(gè)細(xì)菌細(xì)胞》實(shí)驗(yàn)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(xì)胞。是一種原核微生物,內(nèi)部結(jié)構(gòu)很簡單，基因組僅有一百多萬堿基對，遠(yuǎn)小于真核生物基因組十億級(jí)的堿基數(shù)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(tuán)隊(duì)合成了長達(dá)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(shí)驗(yàn)。如果僅僅從技術(shù)上來說，Venter做了一個(gè)無懈可擊的實(shí)驗(yàn)，“人造生命”思路和流程都做得無懈可擊。三個(gè)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(shí)驗(yàn)確定維持其生命周期的最小基因組，并加上4個(gè)“水印基因”作為標(biāo)記。用計(jì)算機(jī)精確計(jì)算需要合成DNA分子序列，并用化學(xué)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(xué)合成”的關(guān)鍵。Venter用化學(xué)方法合成了一千多個(gè)約1kb的DNA片段，作為這次組裝的基本材料。組裝：因?yàn)楹铣缮飳W(xué)技術(shù)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(zhǔn)確的連成超過1000kb的片段。移植：

Venter等把這個(gè)合成基因組移植到不含限制性酶切系統(tǒng)的山羊支原體中，基因組能使用后者的酶系統(tǒng)進(jìn)行自我復(fù)制，經(jīng)過多代繁殖后，長成的菌落已經(jīng)純粹由蕈狀支原體組成。Venter：“創(chuàng)造了一個(gè)計(jì)算機(jī)為父母的生命”JCVI：將8個(gè)由60個(gè)核苷酸組成的DNA片段，

首次人工合成實(shí)驗(yàn)老鼠的線粒體基因組使用8個(gè)只含有60個(gè)核苷酸的DNA片段，讓它們同酶和化學(xué)試劑的混合物相結(jié)合，在50℃下孵化1小時(shí)，5天內(nèi)合成出了實(shí)驗(yàn)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(xì)胞內(nèi)的異常。

用途：生物能源、生物除污…Venter下一步的計(jì)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(zhuǎn)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(jī)。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(fù))NewdevicesandassemblytechnologiesNanofabricationofmicroandmacromaterialsEnergyproductionandstorage(能量產(chǎn)生與儲(chǔ)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(chuàng)建仿地球的生存環(huán)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(chuàng)造的東西，我無法理解——只有通過創(chuàng)造才能理解。不能理解的東西，我無法創(chuàng)造。WhatIcannotcreateIdonotunderstand.——美國物理學(xué)家理查德·費(fèi)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