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Team:UCSF/Synthetic Chromatin Design
From 2008.igem.org
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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 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.
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. 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
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