Team:Tianjin/Data

From 2012.igem.org

(Difference between revisions)
(→Agarose Gel Electrophoresis)
Line 250: Line 250:
[[file:TJU12-data11.JPG|thumb|left|9.16 yeast cPCR 2]][[file:TJU12-data12.JPG|thumb|none|8.01 16s GFP & RFP]]
[[file:TJU12-data11.JPG|thumb|left|9.16 yeast cPCR 2]][[file:TJU12-data12.JPG|thumb|none|8.01 16s GFP & RFP]]
 +
 +
===The Difficulty of Large Fragments Assembly===
 +
The general digestion connection will leave scar and will be limited by the specific cleavage sequences.
 +
[[file:TJU2012-Proj-LMR-fig-2.png|thumb|150px|center|'''Figure 2.''' Enzyme digestion (from the website of dnaQ]]
 +
Long PCR fragment will suffer the decline of success rate and distortion, etc.
 +
[[file:TJU2012-Proj-LMR-fig-3.png|thumb|500px|center|'''Figure 3.''' PCR Recombinant & PCR Machine (from the website of dnaQ]]
 +
However, the construction of some large fragments cannot be avoided, so the development of a low-cost, simple operation, good fidelity, a little limiting factor large DNA fragment assembly method is particularly important.
 +
 +
=Yeast Assembler=
 +
===History===
 +
Yeast Assembler is based on in vivo homologous recombination in yeast. As for its high efficiency and ease to work with, in vivo homologous recombination in yeast has been widely used for gene cloning, plasmid construction and library creation. In the early of 2008, Zengyi Shao from University of lllinois at Urbana-Champaign, Urbana, used such a method to construct biochemical pathways. Such a method, for its high efficiency in assembling multiple genes, received great popularity since its appearance.
 +
 +
===Principles===
 +
One step assembly into a vector.
 +
[[file:TJU2012-Proj-LMR-fig-4.png|thumb|500px|center|'''Figure 4.''' Principles of Yeast assembler (from "DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways")]]
 +
When parts are transformed all parts into Yeast, homologous recombination occurs at the site “x”, and then all little parts are integrated into a vector.
 +
 +
===Advantages and Disadvantages===
 +
Compared with other methods，the “Yeast assembler” are more efficient and useful for large gene assemble.
 +
[[file:TJU2012-Proj-LMR-fig-5.png|thumb|500px|center|'''Figure 5.''' Advantages and disadvantages of three assemble methods (from "DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways")]]
 +
 +
===Completely Synthesizing the Genome of Mycoplasma Genitalium using Yeast Assembler===
 +
In 2008, Gibson from the J. Craig Venter Institute, published an article “one-step assembly in Yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome”. In the article, the author transformed 25 overlapping DNA fragments into Yeast, homologous recombination occurs, and then the whole genome is synthesized.
 +
[[file:TJU2012-Proj-LMR-fig-6.png|thumb|500px|center|'''Figure 6.''' Synthetic Mycoplasma genitalium genome (from "One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic ''Mycoplasma genitalium'' genome")]]
 +
Construction of a synthetic M. genitalium genome in yeast. Yeast cells were transformed with 25 different overlapping A-series DNA segments (blue arrows; ~17 kb to35 kb each) composing the M. genitalium genome. To assemble these into a complete genome, a single yeast cell (tan) must take up at least one representative of the 25 different DNA fragments and incorporate them in the nucleus (yellow), where homologous recombination occurs. This assembled genome, called JCVI-1.1, is 590,011 bp, including the vector sequence (red triangle) shown internal to A86 – 89.
{{:Team:Tianjin/footer}}
{{:Team:Tianjin/footer}}

Revision as of 13:18, 24 October 2012

BioBrick

Favorite Tianjin 2012 iGEM Team Parts

    Name Type Description Designer Length
1 W BBa_K821001 Reporter RFP Reporter with o-RBS Qinfeng Wu, Bin Jia 1059
2 W BBa_K821002 Coding Orthogonal 16S DNA sequence from rrnB Qinfeng Wu, Bin Jia 1894
3 W BBa_K821012 Generator PBad(promoter)+orthogonal 16S RNA Jinlai Zhang, Dongchang Qin 3226

Tianjin 2012 iGEM Team Parts Sandbox

    Name Type Description Designer Length
1 W BBa_K821003 Coding 16S DNA sequence from rrnB Jinlai Zhang, Dongchang Qin 1894
2 W BBa_K821004 Regulatory PBad Promoter Jinlai Zhang, Dongchang Qin 1324
3 W BBa_K821005 Reporter CmR label from pKD3 Qinfeng Wu, Bin Jia 1014
4 W BBa_K821006 Project KanR label from pKD3 Jinlai Zhang, Dongchang Qin 1304
5 W BBa_K821007 Composite PL(promoter)+orthogonal 16S RNA Qinfeng Wu, Bin Jia 1957
6 W BBa_K821008 Composite PLtet+orthogonal 16S RNA Jinlai Zhang, Dongchang Qin 1956
7 W BBa_K821009 RBS Orthogonal RBS 1 Qinfeng Wu, Bin Jia 6
8 W BBa_K821010 Composite PLacI+Native RBS+RFP+Native RBS+GFP+Terminator Qinfeng Wu, Bin Jia 1833
9 W BBa_K821003 Composite PLacI+Orthogonal RBS+RFP+Native RBS+GFP+Terminator Jinlai Zhang, Dongchang Qin 1827

Agarose Gel Electrophoresis

7.14 t7 rbs' crtE
7.31 16s' RFP-GFP AMP


8.03 pSB1C3 K3 Digestion
8.16 16s' mutate to remove EcoRI


8.17 16S' pSB1C3
8.17 Mutate the RBS of Amp using PYES2


8.17 Yeast Assembly using pSB3C5R & pSB3K3
8.18 pBad-16S' remove EcorI, PSB1C3


9.2 6I 6G Gel extraction and verification
9.2 6I,6G P16S Gel extraction and verification


9.2 P16S digestion
9.16 yeast cPCR 1


9.16 yeast cPCR 2
8.01 16s GFP & RFP


The Difficulty of Large Fragments Assembly

The general digestion connection will leave scar and will be limited by the specific cleavage sequences.

Figure 2. Enzyme digestion (from the website of dnaQ

Long PCR fragment will suffer the decline of success rate and distortion, etc.

Figure 3. PCR Recombinant & PCR Machine (from the website of dnaQ

However, the construction of some large fragments cannot be avoided, so the development of a low-cost, simple operation, good fidelity, a little limiting factor large DNA fragment assembly method is particularly important.

Yeast Assembler

History

Yeast Assembler is based on in vivo homologous recombination in yeast. As for its high efficiency and ease to work with, in vivo homologous recombination in yeast has been widely used for gene cloning, plasmid construction and library creation. In the early of 2008, Zengyi Shao from University of lllinois at Urbana-Champaign, Urbana, used such a method to construct biochemical pathways. Such a method, for its high efficiency in assembling multiple genes, received great popularity since its appearance.

Principles

One step assembly into a vector.

Figure 4. Principles of Yeast assembler (from "DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways")

When parts are transformed all parts into Yeast, homologous recombination occurs at the site “x”, and then all little parts are integrated into a vector.

Advantages and Disadvantages

Compared with other methods，the “Yeast assembler” are more efficient and useful for large gene assemble.

Figure 5. Advantages and disadvantages of three assemble methods (from "DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways")

Completely Synthesizing the Genome of Mycoplasma Genitalium using Yeast Assembler

In 2008, Gibson from the J. Craig Venter Institute, published an article “one-step assembly in Yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome”. In the article, the author transformed 25 overlapping DNA fragments into Yeast, homologous recombination occurs, and then the whole genome is synthesized.

Figure 6. Synthetic Mycoplasma genitalium genome (from "One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome")

Construction of a synthetic M. genitalium genome in yeast. Yeast cells were transformed with 25 different overlapping A-series DNA segments (blue arrows; ~17 kb to35 kb each) composing the M. genitalium genome. To assemble these into a complete genome, a single yeast cell (tan) must take up at least one representative of the 25 different DNA fragments and incorporate them in the nucleus (yellow), where homologous recombination occurs. This assembled genome, called JCVI-1.1, is 590,011 bp, including the vector sequence (red triangle) shown internal to A86 – 89.


Retrieved from "http://2012.igem.org/Team:Tianjin/Data"