Team:UCSF/Synthetic Chromatin Design

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     <h2 align="justify">The Yeast Native System</h2>
     <h2 align="justify">The Yeast Native System</h2>
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     <p align="justify">The specifics of heterochromatin (e.g. histone tail mark, binding proteins etc.) varies by organism, but there seems to be a conserved general mode of spreading. First, a histone modifier (pink half circle) is recruited to a site of initiation, where it makes local histone tail modifications. These modified tails serve as sites of recruitment for binding proteins (yellow hexagons) which then recruit more histone modifiers. Thus, positive feedback at the level of histone tail modification propagates silencing outward. In S. cerevisiae, the histone modifier is Sir2 (Silent Information Regulator 2), a histone de-acetylase that primarily targets H4K16. This mark is bound by Sir3 and Sir4, which then recruit more Sir2. </p>
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     <p align="justify">The specifics of heterochromatin (e.g. histone tail mark, binding proteins etc.) varies by organism, but there seems to be a conserved mode of spreading. First, a histone modifier (pink half circle) is recruited to a site of initiation, where it makes local histone tail modifications. These modified tails serve as sites of recruitment for binding proteins (yellow hexagons) which then recruit more histone modifiers. Thus, positive feedback at the level of histone tail modification propagates silencing outward. In S. cerevisiae, the histone modifier is Sir2 (Silent Information Regulator 2), a histone de-acetylase that primarily targets H4K16. This mark is bound by Sir3 and Sir4, which then recruit more Sir2. </p>
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    <br></br>
     <p align="center"><img src="https://static.igem.org/mediawiki/2008/b/bc/Native_System.png" width="450" height="450" /></p>
     <p align="center"><img src="https://static.igem.org/mediawiki/2008/b/bc/Native_System.png" width="450" height="450" /></p>
     <p align="justify"></p>
     <p align="justify"></p>
     <br><br></br></br>
     <br><br></br></br>
     <h2 align="justify">Design of our Yeast Synthetic Chromatin System</h2>
     <h2 align="justify">Design of our Yeast Synthetic Chromatin System</h2>
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     <p align="justify">Our first goal was to direct silencing to a new genomic locus (A). To provide a "landing pad" for the synthetic initiation of silencing, we inserted LexA operators at the site of interest (B). To target Sir2 to this location, we fused the LexA DNA binding domain to its amino terminus, with expression controlled by a galactose-inducible promoter (C). LexA-Sir2 binding to the operator sites leads to local histone de-acetylation. Thee marks are bound by Sir3/Sir4, and more Sir2 is recruited, spreading silencing outward across a target gene (D). To assess oour silencing constructs by flow cytometry (highly quantitative single cell analysis) we used a reporter gene, typically GFP </p>
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     <p align="justify">Our first goal was to direct silencing to a new genomic locus (A). To provide a "landing pad" for the synthetic initiation of silencing, we inserted LexA operators at the site of interest (B). To target Sir2 to this location, we fused the LexA DNA binding domain to its amino terminus, with expression controlled by a galactose-inducible promoter (C). </p>
     <table width="200" border="0" cellpadding="3">
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     <p align="justify">text here</p>
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     <p align="justify">LexA-Sir2 binding to the operator sites leads to local histone de-acetylation (D). These marked histone tails are bound by Sir3/Sir4, and more Sir2 is recruited, spreading silencing outward across a target gene. </p>
     <p align="justify">&nbsp;</p>
     <p align="justify">&nbsp;</p>
     <p align="justify"><img src="https://static.igem.org/mediawiki/2008/6/67/Our_system_D.jpg" width="400" height="199" /></p>
     <p align="justify"><img src="https://static.igem.org/mediawiki/2008/6/67/Our_system_D.jpg" width="400" height="199" /></p>
     <p align="justify">&nbsp;</p>
     <p align="justify">&nbsp;</p>
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     <p align="justify">text here</p>
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     <p align="justify">To assess our silencing constructs by flow cytometry, we used a reporter gene, typically GFP (E).</p>
     <p align="justify"><img src="https://static.igem.org/mediawiki/2008/3/37/Our_system_E.png" width="400" height="124" /></p>
     <p align="justify"><img src="https://static.igem.org/mediawiki/2008/3/37/Our_system_E.png" width="400" height="124" /></p>
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     <p align="justify">text here</p>
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     <p align="justify">Spreading of silencing across GFP leads to a loss of fluorescence. </p>
     <p align="justify">&nbsp;</p>
     <p align="justify">&nbsp;</p>
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    <p align="justify">&nbsp;</p>
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    <p align="justify">Animation  by Leeza showing the construction of our synthetic silencing system</p>
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    <object width="425" height="350"> <param name="movie" value="http://www.youtube.com/v/tWH1t2205ng"> </param> <embed src="http://www.youtube.com/v/tWH1t2205ng" type="application/x-shockwave-flash" width="425" height="350"> </embed> </object>
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    <p align="justify">&nbsp;</p>
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    <p align="justify">Here is a more artistic interpretation of silencing by Jacinto Chen (this animation is still under construction)</p>
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    <object width="425" height="350"> <param name="movie" value="http://www.youtube.com/v/1YS2VRoerE8"> </param> <embed src="http://www.youtube.com/v/1YS2VRoerE8" type="application/x-shockwave-flash" width="425" height="350"> </embed> </object>
     <p align="justify">&nbsp;</p>
     <p align="justify">&nbsp;</p>
     <p align="right"><a href="https://2008.igem.org/Team:UCSF/Synthetic_Chromatin_Properties">Next...</a></p>
     <p align="right"><a href="https://2008.igem.org/Team:UCSF/Synthetic_Chromatin_Properties">Next...</a></p>

Latest revision as of 22:43, 29 October 2008

Untitled Document

Synthetic Chromatin Bit

 

The Yeast Native System

The specifics of heterochromatin (e.g. histone tail mark, binding proteins etc.) varies by organism, but there seems to be a conserved mode of spreading. First, a histone modifier (pink half circle) is recruited to a site of initiation, where it makes local histone tail modifications. These modified tails serve as sites of recruitment for binding proteins (yellow hexagons) which then recruit more histone modifiers. Thus, positive feedback at the level of histone tail modification propagates silencing outward. In S. cerevisiae, the histone modifier is Sir2 (Silent Information Regulator 2), a histone de-acetylase that primarily targets H4K16. This mark is bound by Sir3 and Sir4, which then recruit more Sir2.







Design of our Yeast Synthetic Chromatin System

Our first goal was to direct silencing to a new genomic locus (A). To provide a "landing pad" for the synthetic initiation of silencing, we inserted LexA operators at the site of interest (B). To target Sir2 to this location, we fused the LexA DNA binding domain to its amino terminus, with expression controlled by a galactose-inducible promoter (C).

LexA-Sir2 binding to the operator sites leads to local histone de-acetylation (D). These marked histone tails are bound by Sir3/Sir4, and more Sir2 is recruited, spreading silencing outward across a target gene.

 

 

To assess our silencing constructs by flow cytometry, we used a reporter gene, typically GFP (E).

Spreading of silencing across GFP leads to a loss of fluorescence.

 

 

Animation by Leeza showing the construction of our synthetic silencing system

 

Here is a more artistic interpretation of silencing by Jacinto Chen (this animation is still under construction)

 

Next...

 


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