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Team:UCSF/Synthetic Chromatin Properties
From 2008.igem.org
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<p align="justify">Transcriptional activators and repressors, the mainstays of current synthetic genetic circuits, work at the level of the promoter by affecting the recruitment of the basal transcription machinery. The eukaryotic cell, however, has a more potent mechanism to regulate gene expression: chromatin (gene silencing). Chromatin-based gene regulation has a number of interesting properties in vivo. We reasoned that it might be a powerful tool for synthetic biology. Therefore, we spent the summer developing a synthetic chromatin system, and demonstrating its unique features .</p><br><br></br></br> | <p align="justify">Transcriptional activators and repressors, the mainstays of current synthetic genetic circuits, work at the level of the promoter by affecting the recruitment of the basal transcription machinery. The eukaryotic cell, however, has a more potent mechanism to regulate gene expression: chromatin (gene silencing). Chromatin-based gene regulation has a number of interesting properties in vivo. We reasoned that it might be a powerful tool for synthetic biology. Therefore, we spent the summer developing a synthetic chromatin system, and demonstrating its unique features .</p><br><br></br></br> | ||
<h2 align="justify">Analysis of our Data</h2> | <h2 align="justify">Analysis of our Data</h2> | ||
| - | <p align="justify">Single-cell analysis was done using flow-cytometry. | + | <p align="justify">Single-cell analysis was done using flow-cytometry. Example cells positive for GFP are seen in the microscope images on the lower right panel and their predicted population distribution is shown in the green curve in the ficticious graph in the left (below).</p><br></br> |
<p align="center"><img src="https://static.igem.org/mediawiki/2008/2/2b/Analysis_data.jpg" width="750" height="458" /></p> | <p align="center"><img src="https://static.igem.org/mediawiki/2008/2/2b/Analysis_data.jpg" width="750" height="458" /></p> | ||
<p align="justify"> </p><br><br></br></br> | <p align="justify"> </p><br><br></br></br> | ||
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<p align="justify"> </p> | <p align="justify"> </p> | ||
<h3 align="justify">1. Targeting of Sir2 leads to complete silencing of reporter</h3> | <h3 align="justify">1. Targeting of Sir2 leads to complete silencing of reporter</h3> | ||
| - | <p align="justify">In the eukaryotic cell, heterochromatin blocks gene expression completely. We targeted the silencing machinery to a transgenic locus and monitored reporter expression | + | <p align="justify">In the eukaryotic cell, heterochromatin blocks gene expression completely. We targeted the silencing machinery to a transgenic locus and monitored reporter expression. </p> |
<p align="center"><img src="https://static.igem.org/mediawiki/2008/d/dc/Minusgalactose.png" width="550" height="159" /></p> | <p align="center"><img src="https://static.igem.org/mediawiki/2008/d/dc/Minusgalactose.png" width="550" height="159" /></p> | ||
<p align="center"><img src="https://static.igem.org/mediawiki/2008/5/57/Plusgalactose.png" width="550" height="217" /></p> | <p align="center"><img src="https://static.igem.org/mediawiki/2008/5/57/Plusgalactose.png" width="550" height="217" /></p> | ||
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<p align="justify"> </p> | <p align="justify"> </p> | ||
<p align="center"><img src="https://static.igem.org/mediawiki/2008/4/45/Galactose_R.jpg" width="500" height="359" /></p> | <p align="center"><img src="https://static.igem.org/mediawiki/2008/4/45/Galactose_R.jpg" width="500" height="359" /></p> | ||
| - | <p align="justify"></p> | + | <p align="justify">After the addition of galactose (inducing LexA-Sir2 expression), we observed complete silencing of the GFP reporter. In this case, a medium constitutive promoter (Cyc1P) was used, but similar results were obtained for other promoters (e.g. Fig1 P, see below).</p> |
<h3 align="justify">2. Dominant over Transcription Factors</h3> | <h3 align="justify">2. Dominant over Transcription Factors</h3> | ||
<blockquote> | <blockquote> | ||
Revision as of 00:12, 30 October 2008
Design of our System (previous)
Synthetic Chromatin Bit (Part II)
Why use Chromatin as a Tool for Synthetic Biology?
Transcriptional activators and repressors, the mainstays of current synthetic genetic circuits, work at the level of the promoter by affecting the recruitment of the basal transcription machinery. The eukaryotic cell, however, has a more potent mechanism to regulate gene expression: chromatin (gene silencing). Chromatin-based gene regulation has a number of interesting properties in vivo. We reasoned that it might be a powerful tool for synthetic biology. Therefore, we spent the summer developing a synthetic chromatin system, and demonstrating its unique features .
Analysis of our Data
Single-cell analysis was done using flow-cytometry. Example cells positive for GFP are seen in the microscope images on the lower right panel and their predicted population distribution is shown in the green curve in the ficticious graph in the left (below).
The Properties of Our Synthetic Chromatin Bit
We tested our synthetic chromatin bit in a number of ways.
1. Targeting of Sir2 leads to complete silencing of reporter
In the eukaryotic cell, heterochromatin blocks gene expression completely. We targeted the silencing machinery to a transgenic locus and monitored reporter expression.
RESULT 1:
After the addition of galactose (inducing LexA-Sir2 expression), we observed complete silencing of the GFP reporter. In this case, a medium constitutive promoter (Cyc1P) was used, but similar results were obtained for other promoters (e.g. Fig1 P, see below).
2. Dominant over Transcription Factors
Heterochromatin plays a primary role in the differentiation of the cells of higher eukaryotes, and therefore must be resistant to activation by transcription factors. We tested whether our synthetic chromatin bit, once closed (heterochromatin), could be activated. We used a GFP reporter driven by the pheromone-inducible Fig1 promoter. Activation of the reporter gene was completely blocked in cells that were grown in galactose to induce silencing. Indeed, even the basal activity of the Fig1 promoter (compare was blocked by silencing.
RESULT 2:
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3. Regional Silencing
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RESULT 3:
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4. Spread of Silencing
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RESULT 4:
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5. Binary
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RESULT 5:
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6. Memory
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RESULT 6:
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