人工合成基因组.ppt

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1、人工生命artificial lifen n2007200720072007年年年年6 6 6 6月月月月 美国科学家克雷格美国科学家克雷格美国科学家克雷格美国科学家克雷格 文特尔和他领导的研究小组宣布，文特尔和他领导的研究小组宣布，文特尔和他领导的研究小组宣布，文特尔和他领导的研究小组宣布，他们首次实现了完整的基因组在物种间的移植，这一成功为首个他们首次实现了完整的基因组在物种间的移植，这一成功为首个他们首次实现了完整的基因组在物种间的移植，这一成功为首个他们首次实现了完整的基因组在物种间的移植，这一成功为首个人造生命人造生命人造生命人造生命的降生奏响了序曲。的降生奏响了序曲。的降生奏响了序

2、曲。的降生奏响了序曲。文特尔说，这次成功让他向着制造出首个文特尔说，这次成功让他向着制造出首个文特尔说，这次成功让他向着制造出首个文特尔说，这次成功让他向着制造出首个人造生命人造生命人造生命人造生命又迈进一又迈进一又迈进一又迈进一步，他将在几个月内利用人工合成的基因组展开类似的移植试验，步，他将在几个月内利用人工合成的基因组展开类似的移植试验，步，他将在几个月内利用人工合成的基因组展开类似的移植试验，步，他将在几个月内利用人工合成的基因组展开类似的移植试验，实现科研史上零的突破。如果试验成功，文特尔就能宣布他造出实现科研史上零的突破。如果试验成功，文特尔就能宣布他造出实现科研史上零的突破。如果

3、试验成功，文特尔就能宣布他造出实现科研史上零的突破。如果试验成功，文特尔就能宣布他造出全球第一个全球第一个全球第一个全球第一个合成生命合成生命合成生命合成生命形式。形式。形式。形式。n n2010201020102010年年年年5 5 5 5月月月月20202020日日日日 J.Craig Venter Institute(JCVI),a not-J.Craig Venter Institute(JCVI),a not-J.Craig Venter Institute(JCVI),a not-J.Craig Venter Institute(JCVI),a not-for-profit gen

4、omic research organization,published for-profit genomic research organization,published for-profit genomic research organization,published for-profit genomic research organization,published results today describing the successful construction of results today describing the successful construction o

5、f results today describing the successful construction of results today describing the successful construction of the first self-replicating,synthetic bacterial cell.The the first self-replicating,synthetic bacterial cell.The the first self-replicating,synthetic bacterial cell.The the first self-rep

6、licating,synthetic bacterial cell.The team synthesized the team synthesized the team synthesized the team synthesized the 1.08 million base pair(1080kb)1.08 million base pair(1080kb)1.08 million base pair(1080kb)1.08 million base pair(1080kb)chromosome of a modified chromosome of a modified chromoso

7、me of a modified chromosome of a modified Mycoplasma mycoides Mycoplasma mycoides Mycoplasma mycoides Mycoplasma mycoides genome.The genome.The genome.The genome.The synthetic cell is called synthetic cell is called synthetic cell is called synthetic cell is called Mycoplasma mycoidesMycoplasma myco

8、idesMycoplasma mycoidesMycoplasma mycoides JCVI-syn1.0 JCVI-syn1.0 JCVI-syn1.0 JCVI-syn1.0 and is the proof of principle that genomes can be and is the proof of principle that genomes can be and is the proof of principle that genomes can be and is the proof of principle that genomes can be designed

9、in the computer,chemically made in the designed in the computer,chemically made in the designed in the computer,chemically made in the designed in the computer,chemically made in the laboratory and transplanted into a recipient cell to laboratory and transplanted into a recipient cell to laboratory

10、and transplanted into a recipient cell to laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by produce a new self-replicating cell controlled only by produce a new self-replicating cell controlled only by produce a new self-replicating cell cont

11、rolled only by the synthetic genome.the synthetic genome.the synthetic genome.the synthetic genome.n nThis research will be published by Daniel Gibson et al in This research will be published by Daniel Gibson et al in This research will be published by Daniel Gibson et al in This research will be pu

12、blished by Daniel Gibson et al in the May 20th edition of the May 20th edition of the May 20th edition of the May 20th edition of Science ExpressScience ExpressScience ExpressScience Express and will appear and will appear and will appear and will appear in an upcoming print issue of in an upcoming

13、print issue of in an upcoming print issue of in an upcoming print issue of ScienceScienceScienceScience.Genome transplantation Genome transplantation in bacteria:changing one in bacteria:changing one species to anotherspecies to another天然完整基因组种间转移天然完整基因组种间转移天然完整基因组种间转移天然完整基因组种间转移:As a step toward pr

14、opagation of As a step toward propagation of synthetic genomes,we completely synthetic genomes,we completely replaced the genome of a bacterial replaced the genome of a bacterial cell with one from another species by cell with one from another species by transplanting a whole genome as transplanting

15、 a whole genome as naked DNA.Intact genomic DNA naked DNA.Intact genomic DNA from from Mycoplasma mycoidesMycoplasma mycoides（蕈状支蕈状支蕈状支蕈状支原体原体原体原体 ）large colony(LC),virtually large colony(LC),virtually free of protein,was transplanted into free of protein,was transplanted into Mycoplasma capricolumM

16、ycoplasma capricolum（山羊支原体）山羊支原体）山羊支原体）山羊支原体）cells by polyethylene glycol(PEG)-cells by polyethylene glycol(PEG)-mediated transformation.Cells mediated transformation.Cells selected for tetracycline resistance,selected for tetracycline resistance,carried by the carried by the M.mycoidesM.mycoides LC

17、 LC chromosome,contain the complete chromosome,contain the complete donor genome and are free of donor genome and are free of detectable recipient genomic detectable recipient genomic sequences.These cells that result sequences.These cells that result from genome transplantation are from genome tran

18、splantation are phenotypically identical to the M.phenotypically identical to the M.mycoides LC donormycoides LC donor strain as judged strain as judged by several criteria.by several criteria.-Science.2007 Aug 3;317:632-8-Science.2007 Aug 3;317:632-8合成基因组合成基因组-Science论文论文ScienceScience 2 July 2010:

19、Vol.329.no.5987,pp.52-56 2 July 2010:Vol.329.no.5987,pp.52-56 Creation of a Bacterial Cell Controlled by a Chemically Synthesized Creation of a Bacterial Cell Controlled by a Chemically Synthesized GenomeGenomeDaniel G.Gibson,1 John I.GlassDaniel G.Gibson,1 John I.Glass et al et alWe report the desi

20、gn,synthesis,and assembly of the 1.08megabase pair We report the design,synthesis,and assembly of the 1.08megabase pair Mycoplasma mycoidesMycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantat

21、ion into a sequence information and its transplantation into a M.capricolumM.capricolum recipient recipient cell to create new cell to create new M.mycoidesM.mycoides cells that are controlled only by the cells that are controlled only by the synthetic chromosome.The only DNA in the cells is the des

22、igned synthetic chromosome.The only DNA in the cells is the designed synthetic DNA sequence,including watermark sequences and other synthetic DNA sequence,including watermark sequences and other designed gene deletions and polymorphisms,and mutations acquired designed gene deletions and polymorphism

23、s,and mutations acquired during the building process.The new cells have expected phenotypic during the building process.The new cells have expected phenotypic properties and are capable of continuous self-replication.properties and are capable of continuous self-replication.合成合成生命生命的基的基本操本操作程作程序序酵母酵

24、母中克中克隆细隆细菌全菌全基因基因组策组策略略Nucleic Acids Research,2010,Vol.38,No.8酵母中克隆支原体全基因组酵母中克隆支原体全基因组Nucleic Acids Research,2010,Vol.38,No.8合成生命的的关关键键步步骤骤根据解读的蕈状支原体（根据解读的蕈状支原体（根据解读的蕈状支原体（根据解读的蕈状支原体（M.mycoidesM.mycoidesM.mycoidesM.mycoides）基因组）基因组）基因组）基因组DNADNADNADNA顺序，文特尔团队设计顺序，文特尔团队设计顺序，文特尔团队设计顺序，文特尔团队设计了了了了10781

25、07810781078个个个个DNADNADNADNA卡盒（卡盒（卡盒（卡盒（cassettecassettecassettecassette）,每个每个每个每个DNADNADNADNA卡盒长卡盒长卡盒长卡盒长1080bp1080bp1080bp1080bp，其末端有，其末端有，其末端有，其末端有80bp80bp80bp80bp的顺序重叠的顺序重叠的顺序重叠的顺序重叠.这些这些这些这些DNADNADNADNA卡盒由卡盒由卡盒由卡盒由JCVIJCVIJCVIJCVI指定的公司指定的公司指定的公司指定的公司lue Heron Biotechnologylue Heron Biotechnology

26、lue Heron Biotechnologylue Heron Biotechnology合成。随后进行三个操作步骤：第一步，从合成。随后进行三个操作步骤：第一步，从合成。随后进行三个操作步骤：第一步，从合成。随后进行三个操作步骤：第一步，从1078107810781078个个个个DNADNADNADNA卡盒中每次取卡盒中每次取卡盒中每次取卡盒中每次取10101010个构建了个构建了个构建了个构建了109109109109个长个长个长个长10kb10kb10kb10kb的的的的DNADNADNADNA卡盒卡盒卡盒卡盒.第二步，从第二步，从第二步，从第二步，从110110110110个个个个1

27、0kbDNA10kbDNA10kbDNA10kbDNA卡盒中每卡盒中每卡盒中每卡盒中每次取次取次取次取10101010个构建了个构建了个构建了个构建了11111111个长个长个长个长100kb100kb100kb100kb的的的的DNADNADNADNA卡盒卡盒卡盒卡盒.第三步，将第三步，将第三步，将第三步，将11111111个长个长个长个长100kb100kb100kb100kb的的的的DNADNADNADNA卡盒在酵母细胞中彼此连接装配成完整的人工基因组卡盒在酵母细胞中彼此连接装配成完整的人工基因组卡盒在酵母细胞中彼此连接装配成完整的人工基因组卡盒在酵母细胞中彼此连接装配成完整的人工基因组

28、,全长全长全长全长1080kb,1080kb,1080kb,1080kb,以以以以酵母人工染色体（酵母人工染色体（酵母人工染色体（酵母人工染色体（YACYACYACYAC）的形式扩增。）的形式扩增。）的形式扩增。）的形式扩增。在酵母细胞中人工基因组在酵母细胞中人工基因组在酵母细胞中人工基因组在酵母细胞中人工基因组DNADNADNADNA不发生甲基化，当进入受体细菌后将被受体不发生甲基化，当进入受体细菌后将被受体不发生甲基化，当进入受体细菌后将被受体不发生甲基化，当进入受体细菌后将被受体限制酶降解限制酶降解限制酶降解限制酶降解,因此需要体外甲基化加以保护。酵母人工染色体（因此需要体外甲基化加以保

29、护。酵母人工染色体（因此需要体外甲基化加以保护。酵母人工染色体（因此需要体外甲基化加以保护。酵母人工染色体（YACYACYACYAC）可以随同染色体复制扩增。因带有标记基因，可在筛选培养基上稳定可以随同染色体复制扩增。因带有标记基因，可在筛选培养基上稳定可以随同染色体复制扩增。因带有标记基因，可在筛选培养基上稳定可以随同染色体复制扩增。因带有标记基因，可在筛选培养基上稳定遗传。遗传。遗传。遗传。从酵母细胞中分离扩增的人工合成蕈状支原体基因组从酵母细胞中分离扩增的人工合成蕈状支原体基因组从酵母细胞中分离扩增的人工合成蕈状支原体基因组从酵母细胞中分离扩增的人工合成蕈状支原体基因组DNADNADNA

30、DNA，随后将人工合，随后将人工合，随后将人工合，随后将人工合成基因组导入山羊支原体（成基因组导入山羊支原体（成基因组导入山羊支原体（成基因组导入山羊支原体（Mycoplasma capricolumMycoplasma capricolumMycoplasma capricolumMycoplasma capricolum）受体细胞，）受体细胞，）受体细胞，）受体细胞，山羊山羊山羊山羊支原体受体细胞中编码限制性核酸内切酶基因已失活支原体受体细胞中编码限制性核酸内切酶基因已失活支原体受体细胞中编码限制性核酸内切酶基因已失活支原体受体细胞中编码限制性核酸内切酶基因已失活。人工合成的蕈。人工合成的

31、蕈。人工合成的蕈。人工合成的蕈状支原体基因组状支原体基因组状支原体基因组状支原体基因组DNADNADNADNA在山羊支原体受体细胞中可以转录在山羊支原体受体细胞中可以转录在山羊支原体受体细胞中可以转录在山羊支原体受体细胞中可以转录mRNA,mRNA,mRNA,mRNA,并可翻译并可翻译并可翻译并可翻译成蛋白质。转化的山羊支原体受体细胞基因组成蛋白质。转化的山羊支原体受体细胞基因组成蛋白质。转化的山羊支原体受体细胞基因组成蛋白质。转化的山羊支原体受体细胞基因组DNADNADNADNA或者被蕈状支原体限或者被蕈状支原体限或者被蕈状支原体限或者被蕈状支原体限制性核酸内切酶破坏，或者在细胞繁殖后丟失。

32、在筛选培养基上，最制性核酸内切酶破坏，或者在细胞繁殖后丟失。在筛选培养基上，最制性核酸内切酶破坏，或者在细胞繁殖后丟失。在筛选培养基上，最制性核酸内切酶破坏，或者在细胞繁殖后丟失。在筛选培养基上，最后只生长人工合成蕈状支原体基因组的细胞克隆。后只生长人工合成蕈状支原体基因组的细胞克隆。后只生长人工合成蕈状支原体基因组的细胞克隆。后只生长人工合成蕈状支原体基因组的细胞克隆。最初的实验中，人工合成的基因组并没有产上预想的结果，没有细胞生长。最初的实验中，人工合成的基因组并没有产上预想的结果，没有细胞生长。最初的实验中，人工合成的基因组并没有产上预想的结果，没有细胞生长。最初的实验中，人工合成的基因

33、组并没有产上预想的结果，没有细胞生长。为此文特尔团队设计了一种检测合成的每个为此文特尔团队设计了一种检测合成的每个为此文特尔团队设计了一种检测合成的每个为此文特尔团队设计了一种检测合成的每个DNADNADNADNA卡盒中是否存在错误的卡盒中是否存在错误的卡盒中是否存在错误的卡盒中是否存在错误的方法。他们将天然的方法。他们将天然的方法。他们将天然的方法。他们将天然的11111111个个个个100kb100kb100kb100kb的的的的DNADNADNADNA卡盒和人工合成的卡盒和人工合成的卡盒和人工合成的卡盒和人工合成的11111111个个个个100 kb100 kb100 kb100 kb的

34、的的的卡盒进行搭配重组，用于检测人工合成的卡盒进行搭配重组，用于检测人工合成的卡盒进行搭配重组，用于检测人工合成的卡盒进行搭配重组，用于检测人工合成的100kb100kb100kb100kb卡盒中是否存在错误卡盒中是否存在错误卡盒中是否存在错误卡盒中是否存在错误.凡是检测到功能有缺陷的人工卡盒，随即进行凡是检测到功能有缺陷的人工卡盒，随即进行凡是检测到功能有缺陷的人工卡盒，随即进行凡是检测到功能有缺陷的人工卡盒，随即进行DNADNADNADNA测序，找出存在的突测序，找出存在的突测序，找出存在的突测序，找出存在的突变并给予矫正变并给予矫正变并给予矫正变并给予矫正,确保进入人工基因组合成的卡盒完

35、全正确。确保进入人工基因组合成的卡盒完全正确。确保进入人工基因组合成的卡盒完全正确。确保进入人工基因组合成的卡盒完全正确。First Self-Replicating Synthetic Bacterial CellFirst Self-Replicating Synthetic Bacterial CellFirst Self-Replicating Synthetic Bacterial CellFirst Self-Replicating Synthetic Bacterial Cell 20-May-2010 20-May-2010 20-May-2010 20-May-2010 合合

36、成成生生命命操操作作图图解解创造这个可复制的试验性单细胞生物花费了创造这个可复制的试验性单细胞生物花费了4,000万美元万美元 取名 Synthia:人造儿人造儿 合合成成基基因因组组结结构构图图人工合成基因组中的人工合成基因组中的水印水印 The secret amino acid messages contained in watermarks The secret amino acid messages contained in watermarks that were embedded in the worlds first manmade bacterial that were em

37、bedded in the worlds first manmade bacterial genome.genome.NCBINCBI checked into the genetic sequence submitted by Venters checked into the genetic sequence submitted by Venters Institute and found the watermarks hidden in plain sight.Institute and found the watermarks hidden in plain sight.The five

38、 coded messages that will go down in history as The five coded messages that will go down in history as embedded in the first synthetic genome embedded in the first synthetic genome VENTERINSTITVTEVENTERINSTITVTECRAIGVENTERCRAIGVENTERHAMSMITHHAMSMITHCINDIANDCLYDECINDIANDCLYDEGLASSANDCLYDEGLASSANDCLYDE 代表代表5 5个作者个作者:Craig Venter,Hamilton Smith,John Glass,:Craig Venter,Hamilton Smith,John Glass,Clyde Hutchison Clyde Hutchison