Team:Kyoto/GSDD/Abstract

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Revision as of 07:11, 21 October 2009

Project1 -- Genetic Expression Depending on Cell division --

In biotechnology, it is a big challenge to control the temporal gene expression depending on the cellular state. In this GSDD (Gene Switch Depending on Duplication) project, we aimed to design and construct a time controllable gene expression system depending on the number of cell division after transformation. For instance, such a system could be used to engineer the bacteria that penetrate through human cells for therapy; we can remove the bacteria from human body at desired time and cell population. In other words, we could control the life span of bacteria, by expressing a desired gene (e.g., a killer gene) by counting the number of cell division (Figure 1).

Fig.1

To achieve this goal, we came up with the idea that “liner DNA” could be used as “a timer vector “ that expresses a repressor protein depending on the cell division. Liner DNA is replicated incompletely because of “the end replication problem (REFERENCE)”. Therefore, liner DNA becomes shorter and shorter during duplication. After certain period of time, the coding region of a repressor gene will be removed. Thus, the gene (e.g., a killer gene) coded in the other plasmid vector (e.g., a bomb vector) controlled by the repressor will be activated to regulate cell fate (Figure 2).

Fig.2

The problem in this idea is that liner DNA is not stable in cell because of the attack of exonuclease and DNA-repair enzymes. To solve this problem, we engineered the liner DNA by introducing the multiple protein binding sites (e.g., Lac I binding site) at the both end of liner DNA (Figure 2). In this idea, the tight and specific interaction between liner DNA and its binding protein reduces the activity of the exonucleases. Thus, liner DNA becomes shorter and shorter step by step, until the repetitive protein binding sites are lost during duplication. After the removal of the repetitive binding sequences, the exonuclease degrades liner DNA quickly, and therefore the expression of the repressor protein will be repressed. We aimed to construct such a cell division-dependent gene expression system using yeast Saccaromyces cerevisiae. Figure 2 is the outline of this system.

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 ==Project1 -- '''Genetic Expression Depending on Cell division''' -- ==
+In biotechnology, it is a big challenge to control the temporal gene expression depending on the cellular state. In this GSDD (Gene Switch Depending on Duplication) project, we aimed to design and construct a time controllable gene expression system depending on the number of cell division after transformation. For instance, such a system could be used to engineer the bacteria that penetrate through human cells for therapy; we can remove the bacteria from human body at desired time and cell population. In other words, we could control the life span of bacteria, by expressing a desired gene (e.g., a killer gene) by counting the number of cell division (Figure 1).
 [[Image:Kyoto_GEDD_1.png|200px|thumb|Fig.1]]
-'''In biotechnology''', genes in vectors soon express after transformation. We want the genes to express depending on the number of cell division after transformation. Ultimately, we want to make a system that can control freely the generation of genetic expression after transformation.
-If we can create a bacteria that cures human, and we inject it in human body, it has bad influence to human body that the artificial bacteria lives in human body forever.
-However, using in this system, we can create life span of bacteria if a gene that expresses in this system is cytorethal.
-'''To achieve''' our purpose, our idea is using liner DNA as a vector that product repressor. Liner DNA is replicated incompletely because of the end replication problem. So, liner DNA becomes short by duplication. Through the repetition of duplication, Liner DNA becomes shorter and shorter, and ultimately the region of a gene becomes lost and the expression of the gene becomes silent. Product of repressor becomes silent and repression of a gene in the other plasmid vector becomes lost and the gene becomes expressed.
+To achieve this goal, we came up with the idea that “liner DNA” could be used as “a timer vector “ that expresses a repressor protein depending on the cell division. Liner DNA is replicated incompletely because of “the end replication problem (REFERENCE)”. Therefore, liner DNA becomes shorter and shorter during duplication. After certain period of time, the coding region of a repressor gene will be removed. Thus, the gene (e.g., a killer gene) coded in the other plasmid vector (e.g., a bomb vector) controlled by the repressor will be activated to regulate cell fate (Figure 2).
 [[Image:Kyoto_GEDD_2_kai.png|700px|thumb|center|Fig.2]]
-'''The problem''' in this idea is that liner DNA is not stable in cell because of exonuclease　and proteins related to DNA repair. To settle this problem, the end of liner DNA is the repeats of specific protein binding sites. In this idea, specific proteins bind this repeats, and protect liner DNA by exonuclease , and proteins related to DNA repair. So,liner DNA becomes shorter and shorter, and when the repeats of specific protein binding sites
+The problem in this idea is that liner DNA is not stable in cell because of the attack of exonuclease and DNA-repair enzymes. To solve this problem, we engineered the liner DNA by introducing the multiple protein binding sites (e.g., Lac I binding site) at the both end of liner DNA (Figure 2). In this idea, the tight and specific interaction between liner DNA and its binding protein reduces the activity of the exonucleases. Thus, liner DNA becomes shorter and shorter step by step, until the repetitive protein binding sites are lost during duplication. After the removal of the repetitive binding sequences, the exonuclease degrades liner DNA quickly, and therefore the expression of the repressor protein will be repressed. We aimed to construct such a cell division-dependent gene expression system using yeast Saccaromyces cerevisiae. Figure 2 is the outline of this system.
-are lost, exonuclease degrade liner DNA and production of repressor becomes silent.
-Now、we make a plan using E.coli and yeast. Figure 2 is the outline of this system in yeast. The proteins protecting the end of liner DNA is GAL4 in yeast, and LACI in E.coli.
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