文特尔人工合成支原体ppt课件.pptx

Creation of a bacterial cell controlled by a chemically synthesized geonome,team members:Zhang Hao,Chen Lifang,Wang Jinyi, He Xin,By:He Xin,John Craig Venter,By:He Xin,HiIm Synthia,STRATEGY,By:He Xin,Key:express the phenotypes of synthetic chromosome in a receptor cell,Recombine,They are homologous fragments,1000bp,80bp,80bp,80bp,presented by Zhang hao,1.priciple,Recombine,presented by Zhang hao,If two plasmids have the homologous sequence， in the yeast,the plasmids will also be recombined,presented by Zhang hao,presented by Zhang hao,We produced a 1080 bp fragment by assembly chemically synthesized oligonucleotidesWe used YCpMmycl, A strain of M. mycoides as templet. Each fragment was based on YCpMmycl genome,1000bp,80bp,2.Process,Insert the fragment into the vector,Transplate those recombine vectors into yeast,We construct 10 vectors,each of them contained a 1080bp,presented by Zhang hao,A 1080 bp fragment produced by assembly chemically synthesized oligonucleotides,presented by Zhang hao,The reserved 80 bp fragment from the last 1080bp fragment,A 10k bp fragment recombined by 10 1080bp fragments,In the yeast,In the yeast a contained 10k fragment was combined by 10 vectors which contained a 1080bp fragment,presented by Zhang hao,Analysis of 10kb assembly intermediater,Not I and Sbf I double restriction digestion analysis of assembly 341-350. These restriction enzymes release the vector fragments (5.5 and 3.4 kb) from the 10-kb insert. Insert DNA was separated from the vector DNA on a 0.8% E-gel (Invitrogen). M indicates the 1-kb DNA ladder (New England Biolabs; NEB).,The reserved 80 bp fragment from the last 10080bp fragment,A 100k bp fragment recombined by 10 10080bp fragments,In the yeast,In the same way,we constructed the 100kb fragment,presented by Zhang hao,Analysis of 100kb assembly intermediater,Analysis of assembly 501-600 purified from yeast. The 105-kb circles (100-kb insert plus 5-kb vector)were separated from the linear yeast chromosomal DNA on a 1% agarosegel by applying 4.5 V/cm for 3 hours. S indicates the BAC-Tracker supercoiled DNA ladder (Epicentre),presented by Zhang hao,we constructed the whole genome（1000kb） step by step,constructed the whole genome（235） step by step,presented by Zhang hao,Analysis of 235 genome by PCR,presented by Zhang hao,Yeast clone sMmYCp235 (235) produced the 11 PCR products expected for a complete genome assembly. For comparison, the natural genome extracted from yeast (WT, wild type) was also analyzed. PCR products were separated on a 2% E-gel (Invitrogen). L indicatesthe 100-bp ladder (NEB),Analysis of whole 235 genome by restriction enzyme digestion,presented by Zhang hao,Natural (WT) and synthetic (235) M. mycoides genomes were isolated from yeast in agarose plugs. In addition, DNA was purified from the host strain alone (H).Agarose plugs were digested with Asc I or BssH II, and fragments were separated by clamped homogeneous electrical field (CHEF) gel electrophoresis. Restriction fragments corresponding to the correct sizes are indicated by the fragment numbers shown in the down picture.,Transplated the genome into the mycoplasma,presented by Zhang hao,Isolated synthetic 235 genome from yeast,Transplated the genome into M.capricolumA new strain of mycoplasma,3.Semisynthetic genome assembly and transplantation.,mixing natural pieces with synthetic ones M.capricolumOnly one of the 100-kb subassemblies, 811-900, was not viable. single-base pair deletion frameshift in dnaA,reason?,presented by Chen lifang,4.Characterization of the synthetic transplants.,.multiplex PCR.gel analysis with Asc I and BssH II,presented by Chen lifang,4.Characterization of the synthetic transplants.,examined colony morphology by plating cells on SP4 agar plates containing X-gal,Scanning and transmission electron micrographs (EMs),JCVI-syn1.0,WT,presented by Chen lifang,4.Characterization of the synthetic transplants.,Gene sequencing: a complete replacement of the M. capricolum genome,presented by Chen lifang,Discussion, obtaining an error-free genome that could be transplanted into a recipient cell to create a new cell controlled only by the synthetic genome was complicated and requiredmany quality-control steps. Following transplantation and replication on a plate to form a colony (30 divisions or 109-fold dilution), progeny will not contain any protein molecules that were present in the original recipient cell (the DNA software builds its own hardware). If the methods can be generalized, design, synthesis, assembly, and transplantation of synthetic chromosomes will no longer be a barrier to the progress of synthetic biology.,24,presented by Wang Jinyi,Research Significance,Its a giant philosophical change in terms of understanding life at its basic level.The methods will be a very powerful set of tools. it can be used to make vaccine, such as flu vaccine, rhinovirus vaccine, even HIV vaccine.It will be helpful to work on environmental issues. develop new strains of algae that can efficiently capture carbon dioxide and make new hydrocarbons, to make normal gasoline.,25,presented by Wang Jinyi,thanks,