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. Flow cytometry measurements were taken using a BD LSR-II flow cytometer (BD Biosciences). For each sample, 10,000 cells were counted, and GFP fluorescence was measured by exciting at 488 nm with a 100 mW Coherent Sapphire laser. Cells that were 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. 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).

RESULT 1:

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 our chromatin bit, once closed (heterochromatin), could be activated. We used a GFP reporter driven by the pheromone-inducible Fig1 promoter.

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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Higher-Order Systems (next)