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1、合成生物学(Synthetic biology)(概念、原理、应用),马飞,人工染色体(技术),BAC(细菌人工染色体)：Bacteria以细菌作为对象，将DNA片段与质粒重组后转入细菌中繁殖YAC(酵母人工染色体)：Yeast以酵母作为对象PAC(噬菌体人工染色体)：Phagemid以噬菌体作为对象TAC(可转化的细菌人工染色体)MAC(哺乳类人工染色体),合成生物学应运而生,Synthetic Biology,What is Synthetic Biology?,Taking an engineering approach to design and applying it to Biol
2、ogy使用工程策略设计并应用于生物学,What is Synthetic Biology?1.Biology2.Chemistry3.Engineering4.Re-Writing,BiologistsChemistsEngineers“Re-Writers”,“The code is 3.6 billion years old.Its time for a re-write.”-Tom Knight,Biology“Test models by building them”,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
3、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。,Wimmer从装配平均长度为69 bp的寡核苷酸入手，结合了化学合成与无细胞体系的从头合成，用了3 年时间完成了这个划时代的工作。,Venter 实验室发展了合成基因组,X-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
4、是第一个被测序的DNA分子。X-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 X-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
5、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,Schultz 实验室研究向大肠杆菌蛋白质生物合成装置中添入新组份，使之能通过基因生成非天然的氨基酸，结果取得了成功。但是要在真核细胞做到这一点还有难度。2003年，Schultz 实验室报道了一种向酵母加 入非天然氨基酸密码
6、子的方法，成功地向蛋白质中导入了5 种氨基酸。目前，能掺入到蛋白质的非天然氨基酸已有80多种。今后将可以直接向蛋白质导入顺磁标记、金属结合、光敏异构化等的氨基酸，促进蛋白质结构与功能的研究。,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
7、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。,发展方向,维斯目前正在研究另外一群称为“规则系统”的基因，他希望细菌能估计刺激物的距离，并根据距离的改变做出反应。该项研究可用来探测地雷位置(TNT：生物传感器)。,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。
8、,J.Craig Venter：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.Craig Venter 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Mycoplasma mycoides的基因组取代与之关系密切的 Mycoplasma capricolum的基因组C.Lartigue et al.Genome transplantation in bacteria:Changing one species to another Science,June 28,2007.,人类历史上第一
9、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。,文特尔研究小组研制出的这种新型染色体即实验室合成支原体(Mycoplasma laboratorium)，是一种经过简化拼接的生殖支原体(Mycoplasma genitalium)DN
10、A序列，他们将这种合成支原体移植到活细胞中，使之在细胞中起主控作用，变换成一种新的染色体。按照实验计划，最终这个染色体将控制这个细胞并变成一个新的生命形式。这种新单细胞生物体被命名为“合成器”，受381个基因控制，包含56万个碱基对。这些基因是维持细菌生命所必备的，使它能够摄食和繁殖。由于新的生物体是在现存生物体上搭建，其繁殖和新陈代谢仍然依赖原来生物体的胞内机制。从这一角度看，它并非完全意义上的新型生命形式。但这种给特定基因赋予特定任务的观点已被众多生物学家广泛接受。,“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
11、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”。,2009：Venter：Science,把蕈状支原体的基因组加以改造，使它能够终移植到山羊支原体内，形成
12、了一个新的蕈状支原体细胞。这也是今年这篇科研论文的雏形，在国外的科学媒体上曾经引发热烈的讨论。,2010年的重要大事：“人造生命”诞生,John Craig Venter搅乱了(生命)科学界,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
13、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
14、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
15、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,What is Synthetic Biology?,从原理角度来看,Synthetic Biology,Undergraduates in Synthetic Bio.,international Genetically Engineered Machines,http:/,Lego Assembly fo
16、r DNA Parts,http:/parts.mit.edu/registry/index.php/Assembly:Standard_assembly,Self-organized Pattern Formation,What can you make in SB?,Arsenic Detector,脓毒症,砷,Modifying life,Biotechnology Techniques that use living organisms or parts of organisms to produce a variety of products(from medicines to in
17、dustrial enzymes)Genetic Engineering Introduction of genetic changes(add,modify,delete)into an organism to achieve some goalSynthetic Biology Create novel biological functions and tools by modifying or integrating well-characterized biological components(i.e.genes,promoters)into higher order genetic
18、 networks,Synthetic Biology History,1970 First gene synthesized from scratch(alanine tRNA)1978 Nobel prize awarded to Werner Arber,Daniel Nathans and Hamilton Smith for the discovery of restriction enzymes 1978(Boyer at UCSF)A synthetic version of the human insulin gene was constructed and inserted
19、into the bacterium E.coli.1980 Kary Mullis invents PCR1991 Affymetrix chip-based oligonucleotide synthesis2003 First iGEM competition,creation of standardized parts libraries at MIT,Biotechnology 1.0 Research Workflow,1.Concept,2.Collect DNA fragments(PCR,isolation,vendors,etc),6.Transform,7.Test,3.
20、Bench work,5.Verify DNA,4.Sequence,DNA synthesis costs are dropping,For example the bacteria Mycoplasma genitalium has the smallest genome out of all living cells:517 genes over 580 kb.Minimal costs of oligo creation(not including error-checking):Mid 1990s:$1/bp=$580,000Circa 2000:$0.35/bp=$203,0002
21、006:$0.11/bp=$63,800Ambitious prediction of not-too-distant future(Church et al,2004):$0.00005/bp=$29,Synthesis lengths are increasing,Commercial DNA Synthesis Companies,Data Source:Rob Carlson,U of W,Seattle,BioneerSouth Korea,CinnagenTehran,Iran,Takara BiosciencesDalian,China,Inqaba BiotecPretoria
22、,South Africa,FermentasVilnius,Lithuania,Bio S&T,Alpha DNA,BiocorpMontreal,Canada,GENEARTRegensberg,Germany,MWGBangalore,India,Zelinsky InstituteMoscow,Russia,ScinoPharmShan-hua,Taiwan,GenosphereParis,France,BiolegioMalden,Netherlands,AmbionAustin,Texas,BiosearchNovato,California,Bio-SynthesisLewisv
23、ille,Texas,ChemgenesWilmington,Mass.,BioSpringFrankfurt am Main,Germany,BiosourceCamarillo,CA,DharmaconLafaette,Co.,CyberGene ABNovum,Sweden,Cortec DNAKingston,Ontario,CA,EurogentecBelgium,U.K.,DNA TechnologyAarhus,Denmark,Genemed SynthesisS.San Francisco,CA,DNA 2.0Menlo Park,CA,MetabionMunich,Germa
24、ny,MicrosynthBalgach,Switzerland,Japan Bio ServicesJapan,Blue Heron BiotechnologyBothell,WA,GeneworksAdelaide,Australia,Imperial Bio-MedicChandigarh,India,Bioserve BiotechnologiesHyderabad,India,GenelinkHawthorne,NY.,DNA Synthesis(Caruthers method),Error Rate:1%0.9950=0.60300 seconds per step,Microa
25、rray oligonucleotide synthesis,The power of parallelism,Chip-based versus linear synthesis,Oligonucleotides synthesized,Single-stranded fragments of 50-90nucleotides 3-overlapping next fragment by 17 nucleotides(Tm calculated 52-56),Steps 1 to 5 involve multiple rounds of PCR(heating to 95,cooling t
26、o 56,and PCR at 72).Number of rounds depends on number of fragments.Carried out by PCR machine.,Final step of amplification of complete genedriven by use of excess of terminal single-stranded fragments,PCR-based oligo ligation,In theory,the scale of synthesis is unlimited,Biotechnology 2.0 Research
27、Workflow,1.Concept,2.Design/debug/test,4.Design oligos,6.Transform,7.Test,5.Synthesize DNA,3.Run code,What are the implications of DNA synthesis capacity+freedom of information?,The problem:“Dual Use”Research,Dual use research includes life sciences research:With legitimate scientific purpose That m
28、ay be misused to pose a biologic threat to public health and/or national security.,How easy is it to get this technology?,What can we do?,Number of Individuals,Individuals Intent,honorable,dishonorable,Bin Laden Genetics,Inc.,DisgruntledResearcher,Garage Bio-Hacker,BasicResearcher,Risk spectrum,Basi
29、c logic circuits,Borrowing from electrical engineering,Protein Expression Basics,RNA polymerase binds to promoterRNAP transcribes gene into messenger RNARibosome translates messenger RNA into protein,Z,Z Promoter,Z Gene,Protein,Transcription,RNA Polymerase,DNA,Translation,Messenger RNA,Regulation Th
30、rough Repression and Induction,Repressor proteins can bind to the promoter and block the RNA polymerase from performing transcriptionThe DNA site near the promoter recognized by the repressor is called an operatorThe target gene can code for another repression protein enabling regulatory cascades,Z
31、Promoter&Operator,Z Gene,R Gene,R,R,R Promoter,TranscriptionTranslation,DNA Binding,RNA Polymerase,Logic Circuits,Proteins are the wires/signalsPromoters+decay implement the gatesAny finite-state digital circuit can be builtFor example,X or Y Z,X,Y,R1,Z,R1,R1,X,Y,Z,=,gene,gene,gene,Transcription-Bas
32、ed Inverter,Protein concentrations are analogous to electrical current BUT proteins do not function in an isolated system and need to be unique,0,1,1,0,R,R,Z,Simple Inverter Model,R,Operator,Z Gene,Z,R,Cooperativity,Cooperative DNA binding is where the binding of one protein increases the likelihood
33、 of a second protein bindingCooperativity adds more non-linearity to the systemIncreases switching sensitivityImproves robustness to noise,Z Promoter&Operator,Z Gene,R Gene,R,R,R Promoter,TranscriptionTranslation,CooperativeDNA Binding,RNA Polymerase,R,Cooperative Inverter Model,R,R,Operator,Z Gene,
34、Z,R,BioCircuit Computer-Aided Design,SPICE,BioSPICE,steady state,dynamics,BioSPICE:a prototype biocircuit CAD tool simulates protein and chemical concentrations intracellular circuits,intercellular communication single cells,small cell aggregates,Genetic Circuit Elements,inputmRNA,ribosome,promoter,
35、outputmRNA,ribosome,operator,translation,transcription,RNAp,RBS,RBS,A BioSPICE Inverter Simulation,input,output,repressor,promoter,They work in vivo Flip-flop(Gardner&Collins,2000)Ring oscillator(Elowitz&Leibler,2000)However,cells are very complex environmentsCurrent modeling techniques poorly predi
36、ct behavior,“Proof of Concept”Circuits,time(x100 sec),A,C,B,B,_S,_R,A,_R,B,_S,A,time(x100 sec),time(x100 sec),RS-Latch(“flip-flop”),Ring oscillator,Cellular Logic Summary,Current systems are limited to less than a dozen gatesThree inverter ring oscillator(Elowitz,2000)RS latch(Gardner,2000)Inter-cel
37、l communication(Weiss,2001)A natural repressor-based logic technology presents serious scalability issuesScavenging natural repressor proteins is time consumingMatching natural repressor proteins to work together is difficult,Cellular Logic Summary,Sophisticated synthetic biological systems require
38、a scalable cellular logic technology with good cooperativityZinc-finger proteins can be engineered to create many unique proteins relatively easilyZinc-finger proteins can be fused with dimerization domains to increase cooperativityA cellular logic technology of only zinc-finger proteins should hope
39、fully be easier to characterize,Single Zinc-Finger Structure,DNA Three BaseRecognition Region,Zinc Atom,AlphaHelix,TwoBetaSheets,Poly-Finger ZFPs,A.C.Jamieson,J.C.Miller,and C.O.Pabo.Drug discovery with engineered zinc-finger proteins.Nature Reviews Drug Discovery,May 2003,Complex systems,Q:But if w
40、e dont fully understand all the rules of biology,how can we create anything more than basic systems?A:We can press our limits by modularizing and simplifying as much as possible.,Standardization of ComponentsPredictable performanceOff-the-shelfMechanical Engineering(1800s)&the manufacturing revoluti
41、on(e.g.Henry Ford)AbstractionInsulate relevant characteristics from overwhelming detailSimple components that can be used in combinationFrom Physics to Electrical Engineering(1900s)Decoupling Design&FabricationRules insulating design process from details of fabricationEnable parts,device,and system
42、designers to work togetherVLSI electronics(1970s),Enabling Synthetic Biology,Characterization,Catalog input-output characteristics of existing and new parts/devices,Standardization,Physical connectionsFunctional connectionsPerformance,SB works via three layers of abstraction,Devices,Parts,Systems,Ab
43、straction in biology,Devices,Parts,Systems,Barriers,-Technological-Legal-Ethical,Synthetic Biology:Intellectual Property,Relationship of synthetic biology to intellectual property law has been largely unexplored.The relevant research space already contains broad patents on foundational technology.Sy
44、nthetic biology commons?Tools of open source property rights coupled with viral licensing,Synthetic Biology:Intellectual Property,What is patentable and/or copyrightable?Broad biological functions Specific sequences Specific uses Sources of uncertainty in synthetic biology as related to IPR definiti
45、onsWhat are effects of alternate definitions of what is patentable and copyrightable on:Development of field?Efficiency?Justice?,Synthetic Biology:Intellectual Property,Patents on fundamental ideas in synthetic biology Example:A patent on the idea of a biological part:a piece of DNA with specific fu
46、nction that can be combined with another part in a predefined fashion.Such a patent would be impossible to circumvent.It represents a fundamental concept that underpins synthetic biology.See Stanford patent on System and method for simulating operation of biochemical systems.United States Patent 591
47、4891,Synthetic Biology:Intellectual Property,Patents on fundamental biological functions Example:A patent on a genetically-encoded inverter Such a patent would be almost impossible to circumvent because it represents a basic biological function that is of use in a range of synthetic biological syste
48、ms.See US Dept of Health patent on Molecular computing elements,gates and flip-flops.United States Patent 6774222 See Boston University patent on Multi-state genetic oscillator.United States Patent 6737269 See Boston University patent on Bistable genetic toggle switch.United States Patent 6841376 Se
49、e Boston University parent on Adjustable threshold switch.United States Patent 6828140,Synthetic Biology:Intellectual Property,Patents on classes of biological molecules with a particular function Example:A patent on the use of zinc finger proteins to bind a specific sequence of DNA.Such a patent is
50、 not impossible to circumvent because there are other proteins that bind DNA and that could be engineered to bind new sequences.See MIT patent on Poly zinc finger proteins with improved linkers.United States Patent 6903185 See Scripps Research Institute patent on Zinc finger binding domains for GNN.
