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(duì)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(duì)象PAC(噬菌體人工染色體)：Phagemid…以噬菌體作為對(duì)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(jiǎn)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。人工合成脊髓髓灰白質(zhì)炎病病毒cDNA美國(guó)紐約大學(xué)學(xué)Wimmer實(shí)驗(yàn)室于2002年報(bào)道了化學(xué)學(xué)合成脊髓髓灰白質(zhì)炎病病毒cDNA，并用RNA聚合酶將它轉(zhuǎn)轉(zhuǎn)成有感染染活力的病毒毒RNA。開辟了利用已已知基因組序序列，不需要要天然模板，，從化合物單單體合成感染染性病毒的先先河。Wimmer從裝配平均長(zhǎng)長(zhǎng)度為69bp的寡核苷酸入入手，結(jié)合了了化學(xué)合成與與無(wú)細(xì)胞體系系的從頭合成成，用了3年時(shí)間完成了了這個(gè)劃時(shí)代代的工作。Venter實(shí)驗(yàn)室發(fā)展了了合成基因組組ΦX-174噬菌體基因是是單鏈環(huán)狀DNA，是歷史上第第一個(gè)被純化化的DNA分子，也是第第一個(gè)被測(cè)序序的DNA分子。ΦX-174噬菌體對(duì)動(dòng)植植物無(wú)害，是是合適的合成成研究對(duì)象。。美國(guó)Venter實(shí)驗(yàn)室發(fā)展了了合成基因組組的工作，該該實(shí)驗(yàn)室只只用兩周就合合成了ΦX-174噬菌體基因(5,386bp)。Venter實(shí)驗(yàn)室的技術(shù)術(shù)改進(jìn)主要有有：(1)用凝膠來提純純寡核苷酸以以減少污染；；(2)嚴(yán)格控制退火火連接溫度來來防止與不正正確的序列發(fā)發(fā)生連接；；(3)采用聚合酶循循環(huán)裝置來裝裝配連結(jié)產(chǎn)物物。合成生物學(xué)國(guó)國(guó)際會(huì)議2004年6月在美國(guó)麻省省理工學(xué)院舉舉行了第一屆屆合成生物物學(xué)國(guó)際會(huì)議議。會(huì)上除討論了了科學(xué)與技術(shù)術(shù)問題外，，還討論了合合成生物學(xué)當(dāng)當(dāng)前與將來的的生物學(xué)風(fēng)險(xiǎn)險(xiǎn)，有關(guān)倫理理學(xué)問題，以以及知識(shí)產(chǎn)權(quán)權(quán)問題。隨著這個(gè)領(lǐng)域域的發(fā)展，對(duì)對(duì)于合成生物物學(xué)的安全性性的考慮愈來來愈多。現(xiàn)在不僅通過過合成生成病病毒，而且已已經(jīng)可以合成成細(xì)菌。合成生物學(xué)開開辟了設(shè)計(jì)生生命的前景一方面有可能能合成模仿生生命物質(zhì)特點(diǎn)點(diǎn)的人工化學(xué)學(xué)系統(tǒng)；另一一方面也可能能重新設(shè)計(jì)微微生物如Keasling實(shí)驗(yàn)室向大腸腸桿菌中導(dǎo)入入青蒿與酵母母的基因，使使大腸桿菌能能在調(diào)節(jié)下合合成青蒿素，，從而顯示了了有效而價(jià)廉廉的治療瘧疾疾的前景合成生物學(xué)今今后將能生成成自然界不存存在的新的微微生物。應(yīng)用示例Schultz實(shí)驗(yàn)室研究向向大腸桿菌蛋蛋白質(zhì)生物合合成裝置中添添入新組份，，使之能通過過基因生成非非天然的氨基基酸，結(jié)果取取得了成功。。但是要在真真核細(xì)胞做到到這一點(diǎn)還有有難度。2003年，Schultz實(shí)驗(yàn)室報(bào)道了了一種向酵母母加入非天天然氨基酸密密碼子的方法法，成功地向向蛋白質(zhì)中導(dǎo)導(dǎo)入了5種氨基酸。目前，能摻入入到蛋白質(zhì)的的非天然氨基基酸已有80多種。今后將可以直直接向蛋白質(zhì)質(zhì)導(dǎo)入順磁標(biāo)標(biāo)記、金屬結(jié)結(jié)合、光敏異異構(gòu)化等的氨氨基酸，促進(jìn)進(jìn)蛋白質(zhì)結(jié)構(gòu)構(gòu)與功能的研研究。應(yīng)用用示示例例Brenner提出出向向細(xì)細(xì)胞胞DNA中摻摻入入天天然然不不存存在在的的堿堿基基來來發(fā)發(fā)展展人人工工遺遺傳傳系系統(tǒng)統(tǒng),支持持人工工生生命命形形式式。合成成生生物物學(xué)學(xué)也也將將對(duì)對(duì)生生命命起起源源，，其其他他生生命命形形式式的的研研究究作作出出貢貢獻(xiàn)獻(xiàn)。?？刂浦粕壳扒?，，研研究究人人員員正正在在試試圖圖控控制制細(xì)細(xì)胞胞的的行行為為，，研研制制不不同同的的基基因因線線路路—————即特特別別設(shè)設(shè)計(jì)計(jì)的的、、相相互互影影響響的的基基因因。。波士士頓頓大大學(xué)學(xué)生生物物醫(yī)醫(yī)學(xué)學(xué)工工程程師師科科林林斯斯已已研研制制出出一一種種““套套環(huán)環(huán)開開關(guān)關(guān)””，，所所選選擇擇的的細(xì)細(xì)胞胞功功能能可可隨隨意意開開關(guān)關(guān)。。加州州大大學(xué)學(xué)生生物物學(xué)學(xué)和和物物理理學(xué)學(xué)教教授授埃埃羅羅維維茨茨等等人人研研究究出出另另外外一一種種線線路路：：當(dāng)某種特特殊蛋白白質(zhì)含量量發(fā)生變變化時(shí)，，細(xì)胞能能在發(fā)光光狀態(tài)和和非發(fā)光光狀態(tài)之之間轉(zhuǎn)換換，起到到有機(jī)振振蕩器的的作用，，打開了了利用生生物分子子進(jìn)行計(jì)計(jì)算的大大門。維斯和加加州理工工學(xué)院化化學(xué)工程程師阿諾諾爾一起起，采用用“定向向進(jìn)化””的方法法，精細(xì)細(xì)調(diào)整研研制線路路，將基基因網(wǎng)絡(luò)絡(luò)插入細(xì)細(xì)胞內(nèi)，，有選擇擇性地促促進(jìn)細(xì)胞胞生長(zhǎng)。。發(fā)展方向向維斯目前前正在研研究另外外一群稱稱為“規(guī)規(guī)則系統(tǒng)統(tǒng)”的基基因，他他希望細(xì)細(xì)菌能估估計(jì)刺激激物的距距離，并并根據(jù)距距離的改改變做出出反應(yīng)。。該項(xiàng)研究究可用來來探測(cè)地地雷位置置(TNT：生物傳傳感器)。維斯另一一項(xiàng)大膽膽的計(jì)劃劃是為成成年干細(xì)細(xì)胞編程程促進(jìn)某些些干細(xì)胞胞分裂成成骨細(xì)胞胞、肌肉肉細(xì)胞或或軟骨細(xì)細(xì)胞等，，讓細(xì)胞胞去修補(bǔ)補(bǔ)受損的的心臟或或生產(chǎn)出出合成膝膝關(guān)節(jié)。。盡管該工工作尚處處初級(jí)階階段，但但卻是生生物學(xué)調(diào)調(diào)控領(lǐng)域域中重要要的進(jìn)展展。J.CraigVenter：基因組組替換成功利用用基因組組取代技技術(shù)，將將一種細(xì)細(xì)菌改變變?yōu)榱硪灰环N與之之親緣關(guān)關(guān)系較為為緊密的的另一細(xì)細(xì)菌。這這種由J.CraigVenter進(jìn)行的““移植植(transplantation)”技術(shù)，有有望將合合成基因因組插入入細(xì)胞，，用于生生產(chǎn)合成成生命。。用Mycoplasmamycoides的基因組組取代與與之關(guān)系系密切的的Mycoplasmacapricolum的基因組組C.Lartigueetal."Genometransplantationinbacteria:Changingonespeciestoanother"Science,June28,2007.人類歷史史上第一一個(gè)人造造染色體體合成成成功美科學(xué)家家稱“人人造生命命”技術(shù)術(shù)已被掌掌握最具爭(zhēng)議議的美國(guó)國(guó)著名科科學(xué)家克克雷格·文特爾宣宣布，他他的研究究小組已已經(jīng)合成成出人類類歷史上上首個(gè)人人造染色色體，并并有可能能創(chuàng)造出出首個(gè)永永久性生生命形式式，以此此作為應(yīng)應(yīng)對(duì)疾病病和全球球變暖的的潛在手手段。該研究部部分由美美國(guó)能源源部出資資，希望望藉此研研制出新新型環(huán)保保燃料。。由文特特爾召集集，諾貝貝爾醫(yī)學(xué)學(xué)獎(jiǎng)獲得得者漢密密爾頓·史密斯領(lǐng)領(lǐng)導(dǎo)的研研究小組組在這方方面已經(jīng)經(jīng)進(jìn)行了了5年研究。。文特爾已已用化學(xué)學(xué)藥品在在實(shí)驗(yàn)室室中研制制出一種種合成染染色體。。文特爾研研究小組組研制出出的這種種新型染染色體即即實(shí)驗(yàn)室室合成支支原體(Mycoplasmalaboratorium)，是一種經(jīng)經(jīng)過簡(jiǎn)化拼拼接的生殖殖支原體(Mycoplasmagenitalium)DNA序列，他們們將這種合合成支原體體移植到活活細(xì)胞中，，使之在細(xì)細(xì)胞中起主主控作用，，變換成一一種新的染染色體。按照實(shí)驗(yàn)計(jì)計(jì)劃，最終終這個(gè)染色色體將控制制這個(gè)細(xì)胞胞并變成一一個(gè)新的生生命形式。。這種新單細(xì)細(xì)胞生物體體被命名為為“合成器器”，受381個(gè)基因控制制，包含56萬(wàn)個(gè)堿基對(duì)對(duì)。這些基基因是維持持細(xì)菌生命命所必備的的，使它能能夠攝食和和繁殖。由由于新的生生物體是在在現(xiàn)存生物物體上搭建建，其繁殖殖和新陳代代謝仍然依依賴原來生生物體的胞胞內(nèi)機(jī)制。。從這一角度度看，它并并非完全意意義上的新新型生命形形式。但這這種給特定定基因賦予予特定任務(wù)務(wù)的觀點(diǎn)已已被眾多生生物學(xué)家廣廣泛接受。?！斑@是人類類自然科學(xué)學(xué)史上一次次重大進(jìn)步步，顯示人人類正在從從閱讀基因因密碼走向向有能力重重新編寫密密碼，這將將賦予科學(xué)學(xué)家新的能能力，從事事以前從未未做過的研研究?！彼Ｍ@項(xiàng)項(xiàng)突破有助助于發(fā)展新新能源，應(yīng)應(yīng)對(duì)氣候變變化造成的的負(fù)面影響響。如創(chuàng)造造出具有特特殊功能的的新微生物物，可被用用作替代石石油和煤炭炭的綠色燃燃料，或用用來幫助清清除危險(xiǎn)化化學(xué)物質(zhì)或或輻射等；；還可用來來合成能吸吸收過多二二氧化碳的的細(xì)菌，為為解決氣候候變暖貢獻(xiàn)獻(xiàn)力量。然而制造永永久生命形形式的前景景極具爭(zhēng)議議性，有可可能激起道道德、倫理理等方面的的激烈辯論論。加拿大生物物倫理學(xué)組組織ETC團(tuán)體主任帕帕特·穆尼說，文文特爾制造造出了“一一個(gè)基架，，在此基架架上人們幾幾乎可以制制造出任何何東西”，，“它可以以用于研究究新型藥物物，也可以以用于對(duì)人人類產(chǎn)生巨巨大威脅的的生物武器器”。2009：：Venter：Science把蕈狀支原原體的基因因組加以改改造，使它它能夠終移移植到山羊羊支原體內(nèi)內(nèi)，形成了了一個(gè)新的的蕈狀支原原體細(xì)胞。。這也是今年年這篇科研研論文的雛雛形，在國(guó)國(guó)外的科學(xué)學(xué)媒體上曾曾經(jīng)引發(fā)熱熱烈的討論論。2010年年的重要大大事：““人造生生命”誕生生JohnCraigVenter攪亂了(生命)科科學(xué)界《用化學(xué)合成成的基因組組構(gòu)建一個(gè)個(gè)細(xì)菌細(xì)胞胞》實(shí)驗(yàn)對(duì)象：：蕈狀支原原體。支原體是已已知的可以以自由生活活的最小生生物,也是最小的的原核細(xì)胞胞。是一種原核核微生物,內(nèi)部結(jié)構(gòu)很很簡(jiǎn)單，基基因組僅有有一百多萬(wàn)萬(wàn)堿基對(duì)，，遠(yuǎn)小于真真核生物基基因組十億億級(jí)的堿基基數(shù)量，這這也是Venter選擇操作它它的原因。。Venter早在1995年就對(duì)生殖殖支原體測(cè)測(cè)序，并致致力于研究究維持自由由生命的最最小基因組組。在2008年，Venter的團(tuán)隊(duì)合成成了長(zhǎng)達(dá)59萬(wàn)堿基對(duì)的的生殖支原原體基因組組。此后，他們們選擇生長(zhǎng)長(zhǎng)速度更快快的蕈狀支支原體來做做實(shí)驗(yàn)。如果僅僅從從技術(shù)上來來說，Venter做了一個(gè)無(wú)無(wú)懈可擊的的實(shí)驗(yàn)，““人造生命命”思路和和流程都做做得無(wú)懈可可擊。三個(gè)步驟：：合成、組組裝和移植植合成：蕈狀支原體體的基因組組是一條大大片段的DNA分子，序列列是A、T、G、C四種脫氧核核糖核苷酸酸的排列組組合。通過實(shí)驗(yàn)確確定維持其其生命周期期的最小基基因組，并并加上4個(gè)“水印基基因”作為為標(biāo)記。用計(jì)算機(jī)精精確計(jì)算需需要合成DNA分子序列，，并用化學(xué)學(xué)方法合成成A、T、G、C堿基，并使使其按所要要求序列延延伸。這是它被稱稱為“人造造生命”或或者“化學(xué)學(xué)合成”的的關(guān)鍵。Venter用化學(xué)方法法合成了一一千多個(gè)約約1kb的DNA片段，作為為這次組裝裝的基本材材料。組裝：因?yàn)楹铣缮飳W(xué)技術(shù)術(shù)上的局限限，不能直直接合成上上萬(wàn)堿基對(duì)對(duì)的DNA大分子，所所以Venter等人巧妙地地借助啤酒酒酵母和大大腸桿菌的的幫助，把把1Kb的DNA分子有序準(zhǔn)準(zhǔn)確的連成成超過1000kb的片段。移植：Venter等把這個(gè)合合成基因組組移植到不不含限制性性酶切系統(tǒng)統(tǒng)的山羊支支原體中，，基因組能能使用后者者的酶系統(tǒng)統(tǒng)進(jìn)行自我我復(fù)制，經(jīng)經(jīng)過多代繁繁殖后，長(zhǎng)長(zhǎng)成的菌落落已經(jīng)純粹粹由蕈狀支支原體組成成。Venter：“創(chuàng)創(chuàng)造了一個(gè)個(gè)計(jì)算機(jī)為為父母的生生命”JCVI：將8個(gè)由60個(gè)核苷酸組組成的DNA片段，首次人工合合成實(shí)驗(yàn)老老鼠的線粒粒體基因組組使用8個(gè)只含有60個(gè)核苷酸的的DNA片段，讓它它們同酶和和化學(xué)試劑劑的混合物物相結(jié)合，，在50℃下孵化1小時(shí)，5天內(nèi)合成出出了實(shí)驗(yàn)鼠鼠的線粒體體基因組，，得到的基基因組能夠夠糾正具有有線粒體缺缺陷的細(xì)胞胞內(nèi)的異常常。用途：生物物能源、生生物除污……Venter下一步步的計(jì)劃就就是合成某某種海藻基基因組，這這種新型海海藻可以通通過光合作作用把空氣氣中的二氧氧化碳轉(zhuǎn)化化成汽油或或者柴油等等清潔能源源，從而有有效解決目目前的氣候候變化和能能源危機(jī)。。疫苗、藥物物、生物能能源、生物物除污等WhatisSyntheticBiology?——從原理角度度來看SyntheticBiologyUndergraduatesinSyntheticBernationalGenetically

EngineeredMachines/registry/index.php/Main_PageLegoAssemblyforDNAParts/registry/index.php/Assembly:Standard_assemblySelf-organizedPatternFormationWhatcanyoumakeinSB?ArsenicDetector膿毒癥砷L(zhǎng)ectureoverviewWhatwe’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-AidedDesignSPICEBioSPICEsteadystatedynamicsintercellularBioSPICE:aprototypebiocircuitCADtoolsimulatesproteinandchemicalconcentrationsintracellularcircuits,intercellularcommunicationsinglecells,smallcellaggregatesGeneticCircuitElementsinputmRNAribosomepromoteroutputmRNAribosomeoperatortranslationtranscriptionRNApRBSRBSABioSPICEInverterSimulationinputoutputrepressorpromoterTheyworkinvivoFlip-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:IntellectualPropertyPatentsonfundamentalideasinsyntheticbiologyExample:Apatentontheideaofabiologicalpart:apieceofDNAwithspecificfunctionthatcanbecombinedwithanotherpartinapredefinedfashion.Suchapatentwouldbeimpossibletocircumvent.Itrepresentsafundamentalconceptthatunderpinssyntheticbiology.SeeStanfordpatentonSystemandmethodforsimulatingoperationofbiochemicalsystems.UnitedStatesPatent5914891SyntheticBiology:IntellectualPropertyPatentsonfundamentalbiologicalfunctionsExample:Apatentonagenetically-encodedinverterSuchapatentwouldbealmostimpossibletocircumventbecauseitrepresentsabasicbiologicalfunctionthatisofuseinarangeofsyntheticbiologicalsystems.SeeUSDeptofHealthpatentonMolecularcomputingelements,gatesandflip-flops.UnitedStatesPatent6774222SeeBostonUniversitypatentonMulti-stategeneticoscillator.UnitedStatesPatent6737269SeeBostonUniversitypatentonBistablegenetictoggleswitch.UnitedStatesPatent6841376SeeBostonUniversityparentonAdjustablethresholdswitch.UnitedStatesPatent6828140SyntheticBiology:IntellectualPropertyPatentsonclassesofbiologicalmoleculeswithaparticularfunctionExample:ApatentontheuseofzincfingerproteinstobindaspecificsequenceofDNA.SuchapatentisnotimpossibletocircumventbecausethereareotherproteinsthatbindDNAandthatcouldbeengineeredtobindnewsequences.SeeMITpatentonPolyzincfingerproteinswithimprovedlinkers.UnitedStatesPatent6903185SeeScrippsResearchInstitutepatentonZincfingerbindingdomainsforGNN.UnitedStatesPatent6610512SeeSangamoBiosciences,Inc.patentonRegulationofendogenousgeneexpressionincellsusingzincfingerproteins.UnitedStatesPatent6607882SyntheticBiology:IntellectualPropertyPatentonaparticularbiologicalmolecule.Example:Apatentonthesequenceofaparticularproteinthatsenseslightandtransmitsasignalintothecell.Suchapatentwouldlikelybefairlyeasytocircumventbecausethereareprobablyafewaminoacidsthatcouldbechangedintheproteinsuchthatitwoulditwouldstillbefunctionalyetnothavetheexactsamesequenceasspecifiedinthepatent.Thereareexceptionstothisrule:Someproteinsthathavebeensooptimizedforaspecificfunctionthatanymutationinthesequencecanleadtolessfunctionality(e.g.,thepeptidedrugZiconitide).OpencommonsofbiologicalfunctionsOpen-accessbiology?Whenatechnologyisproprietary,boththeabilityandinterestinexamining&troubleshootingproblemsisrestrictedtothosewiththeIPMightopen-accessbiologygenerateahigherqualityproduct?Orwoulditstifleinnovationthroughalackofinterest?ProgrammedOrganisms(編程性物種種)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)生與與儲(chǔ)存)NewbiologicalpathwaysSyntheticBiologyApplicationsMedicalMolecularmedicaldevicesReversalofaging(返老還童)Diseasefighting(抗病)Implantablelivingbatteryformedicaldeviceoutofelectriceelcells.Humansthatphotosynthesize(人類光合成成)SyntheticBiologyApplicationsSensors(傳感器)SmartsensorsUsecellstoread,process,outputinformationDetectarbitrarysubstancesSelf-reproducingchemical/radioactivitysensorsDetectbiotoxinsandencapsulate.flashwhenitdoes.Responsivematerials(e.g.,oillubricantsbydesign/need)ToolstomeasureconcentrationofproteinincellEcosystemdebugger(read/write)IntelligentBiosensors(智能型傳感感器)SyntheticBiologyApplicationsTerraformingCreatinglifeonotherworlds仿地成形（（尤指在科科幻小說中中，在外星星球創(chuàng)建仿仿地球的生生存環(huán)境，，以使人類類能夠生存存）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(wú)法創(chuàng)造造的東西西，我無(wú)無(wú)法理解解——只有通過過創(chuàng)造才才能理解解。不能能理解的的東西，，我無(wú)法法創(chuàng)造。。WhatIcannotcreateIdonotunderstand.———美國(guó)國(guó)物物理理學(xué)學(xué)家家理理查查德德·費(fèi)曼曼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-Incompleteknowledgeofbiology.2-Inherentfunctionaloverlap(partswithmany-someunknown-functions,someofwhicharedetrimentaltothegoalinmind.3-Incompatibilitybetweenparts.4-Partsfunctionalitydependsoncontext.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”tomonitorinr