Team:UT-Tokyo/Project/Inhibition/Background

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このページの概要を、簡単に記述。
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Here we explain the Background of Inhibition without Knockout project.
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== Background ==
== Background ==
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In project H2 E.coli, we tried to improve the formic acid-hydrogen pathway by overexpressing a gene fhlA which controls a step in this pathway on plasmid.
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In project H2 E.coli, we tried to improve the formic acid-hydrogen pathway by overexpressing a gene fhlA which controls a step in this pathway.
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However, E.coli possess a protein called HycA in their genome preventing unrestricted hydrogen synthesis. In order to increase the amount of hydrogen production, it was thought that there was a need for systems that inhibits the action of negative factors such as HycA.
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However, E.coli possess a protein called HycA coded in their genome preventing unrestricted hydrogen synthesis. In order to increase the amount of hydrogen produced, it was thought that there was a need for systems that inhibit the action of negative factors such as HycA.
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In general, in order to suppress the negative factors such as HycA, gene knockout method is used. However, because it is easy to introduce plasmid rather than gene knockout method, we decided to supress the factors by adding Biobrick Parts. Plasmid in which a large number of connecting each DNA sequence that is the binding site of factor was introduced into E.coli as a decoy to bind factors. Thus, the factors binds to the decoy Plasmid and we can reduce the binding to the site to inhibit the function. Additionally, by changing the number of decoy binding sites, transcription can be adjusted stepwise.
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In general, in order to suppress negative factors such as HycA, a gene knockout method is used. However, because it is easier to introduce a plasmid rather than knocking out a gene, we decided to try to suppress these factors by adding Biobrick Parts. A plasmid containing a large number of repeats of the binding site of the factor is introduced into E.coli as a decoy to bind the factors. Thus, the factors bind to the decoy Plasmid and we can reduce the binding to its original binding site to inhibit the function. Additionally, by changing the number of decoy binding sites, transcription can be adjusted stepwise.
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To this end we used LacI, AraC and ArgR whose binding sites are known, and introduced multiple copies of their binding sites into plasmids to examine how the expression levels of genes downstream of these proteins were affected.
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To this end we used LacI and ArgR whose binding sites are known, and introduced multiple copies of their binding sites into plasmids to examine how the expression levels of genes downstream of these proteins were affected.
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Latest revision as of 22:56, 26 September 2012

Inhibition without Knockout: Background

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Here we explain the Background of Inhibition without Knockout project.

Background

In project H2 E.coli, we tried to improve the formic acid-hydrogen pathway by overexpressing a gene fhlA which controls a step in this pathway.

However, E.coli possess a protein called HycA coded in their genome preventing unrestricted hydrogen synthesis. In order to increase the amount of hydrogen produced, it was thought that there was a need for systems that inhibit the action of negative factors such as HycA.

In general, in order to suppress negative factors such as HycA, a gene knockout method is used. However, because it is easier to introduce a plasmid rather than knocking out a gene, we decided to try to suppress these factors by adding Biobrick Parts. A plasmid containing a large number of repeats of the binding site of the factor is introduced into E.coli as a decoy to bind the factors. Thus, the factors bind to the decoy Plasmid and we can reduce the binding to its original binding site to inhibit the function. Additionally, by changing the number of decoy binding sites, transcription can be adjusted stepwise.

To this end we used LacI and ArgR whose binding sites are known, and introduced multiple copies of their binding sites into plasmids to examine how the expression levels of genes downstream of these proteins were affected.