文特尔人工合成支原体

presented by Chen lifang
4.Characterization of the synthetic transplants.
③examined colony
morphology by plating cells on SP4 agar plates containing X-gal
digestion analysis of assembly 341350. 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).
④Scanning and
transmission electron micrographs (EMs)
↓
↓
JCVIsyn1.0
WT
presented by Chen lifang
4.Characterization of the synthetic transplants.
• ⑤Gene sequencing:
3.Semisynthetic genome assembly and transplantation.
• mixing natural pieces with synthetic ones M.capricolum
• Only one of the 100-kb subassemblies, 811-900, was not viable.
presented by Zhang hao
Analysis of whole 235 genome by restriction enzyme digestion
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. presented by Zhang hao
Insert the fragment into the vector
presented by Zhang hao
Transplate those recombine vectors into yeast
presented wenku.baidu.comy Zhang hao
In the yeast a contained 10k fragment was combined by 10 vectors which contained a 1080bp fragment
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
Assemble chromosome in yeast
transplant into a recipient bacterial cell
Key:express the phenotypes of synthetic chromosome in a receptor cell
By:He Xin
1.priciple
presented by Zhang hao
2.Process
We produced a 1080 bp fragment by assembly chemically synthesized oligonucleotides We used YCpMmycl, A strain of M. mycoides as templet. Each fragment was based on YCpMmycl genome
In the yeast
The reserved 80 bp fragment from the last 1080bp fragment
presented by Zhang hao
A 10k bp fragment recombined by 10 1080bp fragments
Analysis of 10kb assembly intermediater Not I and Sbf I double restriction
80bp
They are homologous fragments
1000bp
Recombine
80bp 80bp
presented by Zhang hao
Recombine
presented by Zhang hao
If two plasmids have the homologous sequence， in the yeast,the plasmids will also be recombined
reason?
• single-base pair deletion frameshift in dnaA
presented by Chen lifang
4.Characterization of the synthetic transplants.
• ①.multiplex PCR
• ②.gel analysis with Asc I and BssH II
Creation of a bacterial cell controlled by a chemically synthesized geonome
team members: Zhang Hao,Chen Lifang, Wang Jinyi, He Xin
Content
Introduction Method
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
Discussion
By:He Xin
John Craig Venter
Hi~I’m Synthia
Human Genome
synthetic cell"Synthia."
By:He Xin
STRATEGY
Design DNA sequence in the computer
Synthesis DNA fragment from chemicals
Transplated the genome into the mycoplasma
Isolated synthetic 235 genome from yeast
Transplated the genome into M.capricolum
A new strain of mycoplasma
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)
(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% Egel (Invitrogen). L indicates the 100-bp ladder (NEB)
of synthetic chromosomes will no longer be a barrier to the progress of synthetic
biology.
24
presented by Wang Jinyi
Research Significance
It's 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.
In the same way,we constructed the 100kb fragment
In the yeast
The reserved 80 bp fragment from the last 10080bp fragment
presented by Zhang hao
A 100k bp fragment recombined by 10 10080bp fragments
>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
we constructed the whole genome（1000kb） step by step
presented by Zhang hao
constructed the whole genome（235） step by step
presented by Zhang hao
Analysis of 235 genome by PCR Yeast clone sMmYCp235
1000bp
80bp
presented by Zhang hao
A 1080 bp fragment produced by assembly chemically synthesized oligonucleotides
We construct 10 vectors,each of them contained a 1080bp