IGEM:Peking University/2008/Proposal

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==A Simple Genetic Circuit for In Vivo Evolution in Eukaryotes==
==A Simple Genetic Circuit for In Vivo Evolution in Eukaryotes==
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Evolution could be dissociated into two consecutive steps: random mutation, and natural selection. Whilst its role in life history is well understood and accepted, evolution is also evident across individual life process. In particular, adaptive immunity system adopts evolution strategy to produce antibodies for novel antigens. Artificial evolution method could be a powerful tool for answering scientific questions or engineering novel biological systems. Via systems biology approach, here we present a simple genetic circuit consisting functional elements for random mutation and artificial selection. This circuit may perform in vivo evolution on virtually any genetically encoded targets, with potential applications in academic and industrial contexts.  
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Evolution could be dissociated into two parallel processes: random mutation, and natural selection. Whilst its role in life history is well understood and accepted, evolution is also evident across individual life process. In particular, adaptive immunity system adopts evolution strategy to produce antibodies for novel antigens. Artificial evolution method could be a powerful tool for answering scientific questions or engineering novel biological systems. Via systems biology approach, here we present a simple genetic circuit consisting functional elements for random mutation and artificial selection. This circuit may perform in vivo evolution on virtually any genetically encoded targets, with potential applications in academic and industrial contexts.  
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The gene encoding the core element in adaptive immunity, hAID, is fused with the LexA-DBD domain with flexible linker. The fusion gene hAID-LexA is inserted into a yeast ESC expression cassette at 3’ of tandem LacO elements. The target gene (your favorite gene, yfg) is inserted into a yeast ESC expression cassette, with its 3’UTR containing tandem LexO elements. An inducible promoter response to yfg activity drives expression of His3, LacZ and LacI in cistrone spanned by IRES. These three plasmids together form a genetic circuit for in vivo evolution.  
The gene encoding the core element in adaptive immunity, hAID, is fused with the LexA-DBD domain with flexible linker. The fusion gene hAID-LexA is inserted into a yeast ESC expression cassette at 3’ of tandem LacO elements. The target gene (your favorite gene, yfg) is inserted into a yeast ESC expression cassette, with its 3’UTR containing tandem LexO elements. An inducible promoter response to yfg activity drives expression of His3, LacZ and LacI in cistrone spanned by IRES. These three plasmids together form a genetic circuit for in vivo evolution.  
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We present one example in yeast one hybrid: a mutated Gal4 gene is inserted in the target cassette. The selector cassette contains UAS to drive expression of His3, LacZ and LacI. All three plasmids are transfected into gal4-, his3-, trp2- yeast strain with proper selection tags. Initially, we culture the yeast in complete YPD medium. Defect Gal4 product cannot bind to UAS, hence hAID-LexA is constitutively expressed, recruited to LexO sites of the target, and mutates the Gal4 conding sequence. Once Gal4 mutation is reversed, it binds to UAS to drive LacI expression, which represses hAID-LexA. We plate the yeast into Trp-, His-, Leu-, 3AT+, XGal+ plate and select for the large, blue colonies. Sequencing the colonies then gives us the activated Gal4 gene sequences.  
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For clearness, here we present one very simple example in yeast one hybrid: a mutated Gal4 gene is inserted in the target cassette. The selector cassette contains UAS to drive expression of His3, LacZ and LacI. All three plasmids are transfected or knock-in-ed into gal4-, his3- yeast strain with proper selection tags. Initially, we culture the yeast in complete YPD medium. Defect Gal4 product cannot bind to UAS, hence hAID-LexA is constitutively expressed, recruited to LexO sites of the target, and mutates the Gal4 conding sequence. Once Gal4 mutation is reversed, it binds to UAS to drive LacI expression, which represses hAID-LexA. We plate the yeast into Trp-, His-, Leu-, 3AT+, XGal+ plate and select for the large, blue colonies. Sequencing the colonies then gives us the activated Gal4 gene sequences.  
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 +
<br>
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The power of this system could be best revealed in the above case: since selection is not required for initiation and attenuation of mutagenesis, there is virtually no need to perform selective pressure titration.
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 +
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The system could be readily adopted to in vivo evolution of any kind of protein-protein and protein-nucleic acid interaction: for this we simply adopt a yeast two hybrid-like approach, evolving a functional protein linked to Gal4 AD which could bind to the "bait" protein linked to Gal4 DBD. We could also use the yeast three hybrid system to study RNA-protein interaction, or use yeast one hybrid to evolve specific protein that binds to given promotor.  
The system could be readily adopted to in vivo evolution of any kind of protein-protein and protein-nucleic acid interaction: for this we simply adopt a yeast two hybrid-like approach, evolving a functional protein linked to Gal4 AD which could bind to the "bait" protein linked to Gal4 DBD. We could also use the yeast three hybrid system to study RNA-protein interaction, or use yeast one hybrid to evolve specific protein that binds to given promotor.  
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Yeast shows several advantages in studying eukaryotic process, for it folds and modifies the eukaryotic protein accurately, whilst bacteria does not. Another advantage of yeast is that cell wall blocks intracellular communication between transmembrane proteins, and the selection happens completely within the cell, therefore blocking possible false positives. We plan to screen for extracellular activator of certain eukaryotic transmembrane protein, for example, novel peptidergic ligand for GPCR and antibody-antigen interaction.
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We have been working on further improvements of the mutation system, by improving the enzyme to archieve directed, "hotspot-less", and evenly distributed mutagenesis, by utilizing the overwhelming power of yeast genetics, and by harnessing novel protein chemistry.
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//Last modified: ZY 20080704
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Yeast shows several advantages in studying eukaryotic process, for it folds and modifies the eukaryotic protein accurately, whilst bacteria does not. Another advantage of yeast is that cell wall blocks intracellular communication between transmembrane proteins, and the selection happens completely within the cell, therefore blocking possible false positives. We plan to screen for extracellular activator of certain eukaryotic transmembrane protein, for example, novel peptidergic ligand for GPCR and antibody-antigen interaction.
 
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We have been working on further improvements of the mutation system, by using better enzyme to archieve directed, "hotspot-less", and evenly distributed mutagenesis, and by utilizing the overwhelming power of yeast genetics.
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Current revision

A Simple Genetic Circuit for In Vivo Evolution in Eukaryotes


Evolution could be dissociated into two parallel processes: random mutation, and natural selection. Whilst its role in life history is well understood and accepted, evolution is also evident across individual life process. In particular, adaptive immunity system adopts evolution strategy to produce antibodies for novel antigens. Artificial evolution method could be a powerful tool for answering scientific questions or engineering novel biological systems. Via systems biology approach, here we present a simple genetic circuit consisting functional elements for random mutation and artificial selection. This circuit may perform in vivo evolution on virtually any genetically encoded targets, with potential applications in academic and industrial contexts.

The gene encoding the core element in adaptive immunity, hAID, is fused with the LexA-DBD domain with flexible linker. The fusion gene hAID-LexA is inserted into a yeast ESC expression cassette at 3’ of tandem LacO elements. The target gene (your favorite gene, yfg) is inserted into a yeast ESC expression cassette, with its 3’UTR containing tandem LexO elements. An inducible promoter response to yfg activity drives expression of His3, LacZ and LacI in cistrone spanned by IRES. These three plasmids together form a genetic circuit for in vivo evolution.

For clearness, here we present one very simple example in yeast one hybrid: a mutated Gal4 gene is inserted in the target cassette. The selector cassette contains UAS to drive expression of His3, LacZ and LacI. All three plasmids are transfected or knock-in-ed into gal4-, his3- yeast strain with proper selection tags. Initially, we culture the yeast in complete YPD medium. Defect Gal4 product cannot bind to UAS, hence hAID-LexA is constitutively expressed, recruited to LexO sites of the target, and mutates the Gal4 conding sequence. Once Gal4 mutation is reversed, it binds to UAS to drive LacI expression, which represses hAID-LexA. We plate the yeast into Trp-, His-, Leu-, 3AT+, XGal+ plate and select for the large, blue colonies. Sequencing the colonies then gives us the activated Gal4 gene sequences.

The power of this system could be best revealed in the above case: since selection is not required for initiation and attenuation of mutagenesis, there is virtually no need to perform selective pressure titration.

The system could be readily adopted to in vivo evolution of any kind of protein-protein and protein-nucleic acid interaction: for this we simply adopt a yeast two hybrid-like approach, evolving a functional protein linked to Gal4 AD which could bind to the "bait" protein linked to Gal4 DBD. We could also use the yeast three hybrid system to study RNA-protein interaction, or use yeast one hybrid to evolve specific protein that binds to given promotor.

Yeast shows several advantages in studying eukaryotic process, for it folds and modifies the eukaryotic protein accurately, whilst bacteria does not. Another advantage of yeast is that cell wall blocks intracellular communication between transmembrane proteins, and the selection happens completely within the cell, therefore blocking possible false positives. We plan to screen for extracellular activator of certain eukaryotic transmembrane protein, for example, novel peptidergic ligand for GPCR and antibody-antigen interaction.

We have been working on further improvements of the mutation system, by improving the enzyme to archieve directed, "hotspot-less", and evenly distributed mutagenesis, by utilizing the overwhelming power of yeast genetics, and by harnessing novel protein chemistry.

//Last modified: ZY 20080704


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