"
Team:UPO-Sevilla/Project/Epigenetic Flip Flop/Designing
From 2011.igem.org
| Line 20: | Line 20: | ||
<h2>Compaction module</h2> | <h2>Compaction module</h2> | ||
| - | <p>It is composed by nmt1 (no message in thiamine) | + | <p>It is composed by nmt1 (<em>no message in thiamine</em>) repressible promoter, three alternative proteins (Sir3p, Swi6 and the Chromo-Shadow Domain of Swi6 “CSD” (<a href="http://www.cell.com/current-biology/abstract/S0960-9822(00)00467-X" target="_blank" title=" Cowieson et al, 2000">Cowieson et al, 2000</a>; <a href="http://www.jbc.org/content/early/2011/01/11/jbc.M110.143198" target="_blank" title="Haldar et al, 2011">Haldar et al, 2011</a>), fused to the Tetracycline Repressor protein (tetR), and nmt Terminator. These modules will be expressed from a plasmid containing an independent inducible promoter, pREP41X (<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC309706/pdf/nar00061-0170.pdf" target="_blank" title="Forsburg, 1993">Forsburg, 1993</a>; <a href="http://www.mendeley.com/research/moreno-durn-ribas2000pdf/" target="_blank" title="Moreno et al, 2000">Moreno et al, 2000</a>). |
These fusion proteins or kymeras will bind to specific tetR operator sequences placed upstream of an inducible promoter and downstream of the marker GFP gene, controlled by that promoter. </p> | These fusion proteins or kymeras will bind to specific tetR operator sequences placed upstream of an inducible promoter and downstream of the marker GFP gene, controlled by that promoter. </p> | ||
| + | <div class="center"> | ||
| + | <img width=" 300px" src="https://static.igem.org/mediawiki/2011/7/76/UPOSevillaCompaction_module.jpg" alt="Compaction Modules" /> | ||
| + | </div> | ||
| + | |||
| + | <p class="center"><strong>[Figure 1].</strong> Alternative compaction modules, composed by Pnmt1 promoter, engineered silencing proteins (tetR+Swi6/CSD/Sir3) and nmt1 terminator. Swi6 belongs to <em>S. pombe</em> and Sir3 belongs to <em>Saccharomyces cerevisiae</em>. | ||
| + | </p> | ||
<br/> | <br/> | ||
| Line 27: | Line 33: | ||
<p>Reporter Module contains a GFP inducible expression system, flanked by tet operator sequences. This module will be integrated and expressed from various regions of the S. pombe genome with different heterochromatin contexts, to test it effect on silencing. We expect that once the protein Sir3, Swi6 or CSD binds the targeting tetR operator sequences they will induce the compaction of the promoter, or at least, prevent its leaky activation, because of the spreading of the engineered silencing proteins and it cooperativity. Additionally, there have been introduce two boundaries or chromatin insulators each site of our system. They are transcriptional terminator that act as neutral transcriptionally elements that prevent negative or positive influence from extending across chromatin to a promoter (<a href="http://www.nature.com/embor/journal/v2/n2/abs/embor484.html" target="_blank" title="Fourel et al, 2001">Fourel et al, 2001</a>; <a href="http://www.cell.com/molecular-cell/abstract/S1097-2765(08)00605-9" target="_blank" title="Bushey et al, 2008">Bushey et al, 2008</a>).</p> | <p>Reporter Module contains a GFP inducible expression system, flanked by tet operator sequences. This module will be integrated and expressed from various regions of the S. pombe genome with different heterochromatin contexts, to test it effect on silencing. We expect that once the protein Sir3, Swi6 or CSD binds the targeting tetR operator sequences they will induce the compaction of the promoter, or at least, prevent its leaky activation, because of the spreading of the engineered silencing proteins and it cooperativity. Additionally, there have been introduce two boundaries or chromatin insulators each site of our system. They are transcriptional terminator that act as neutral transcriptionally elements that prevent negative or positive influence from extending across chromatin to a promoter (<a href="http://www.nature.com/embor/journal/v2/n2/abs/embor484.html" target="_blank" title="Fourel et al, 2001">Fourel et al, 2001</a>; <a href="http://www.cell.com/molecular-cell/abstract/S1097-2765(08)00605-9" target="_blank" title="Bushey et al, 2008">Bushey et al, 2008</a>).</p> | ||
| + | <div class="center"> | ||
| + | <img width="400px" src="https://static.igem.org/mediawiki/2011/d/d4/UPOSevillaReporter_module.jpg" alt="Reporter Module" /> | ||
| + | </div> | ||
| + | <p class="center"><strong>[Figure 5]. Reporter module:</strong>. Kan-MX6 (selectable cassette for integration in leu1), Adh1 terminator (upstream insulator ), tetO2 (upstream operator sites), urg1 promoter, GFP marker gene, Adh1 terminator, tetO4 (downstream operator sites), Act1 terminator (downstream insulator).</p> | ||
| + | <br/> | ||
| + | |||
</div> | </div> | ||
Revision as of 00:23, 20 September 2011
Designing
The epigenetic biestable has two main modules, based on Bahler's modules (Bähler et al, 1998; Watt et al, 2008):
Compaction module
It is composed by nmt1 (no message in thiamine) repressible promoter, three alternative proteins (Sir3p, Swi6 and the Chromo-Shadow Domain of Swi6 “CSD” (Cowieson et al, 2000; Haldar et al, 2011), fused to the Tetracycline Repressor protein (tetR), and nmt Terminator. These modules will be expressed from a plasmid containing an independent inducible promoter, pREP41X (Forsburg, 1993; Moreno et al, 2000). These fusion proteins or kymeras will bind to specific tetR operator sequences placed upstream of an inducible promoter and downstream of the marker GFP gene, controlled by that promoter.
[Figure 1]. Alternative compaction modules, composed by Pnmt1 promoter, engineered silencing proteins (tetR+Swi6/CSD/Sir3) and nmt1 terminator. Swi6 belongs to S. pombe and Sir3 belongs to Saccharomyces cerevisiae.
Reporter module
Reporter Module contains a GFP inducible expression system, flanked by tet operator sequences. This module will be integrated and expressed from various regions of the S. pombe genome with different heterochromatin contexts, to test it effect on silencing. We expect that once the protein Sir3, Swi6 or CSD binds the targeting tetR operator sequences they will induce the compaction of the promoter, or at least, prevent its leaky activation, because of the spreading of the engineered silencing proteins and it cooperativity. Additionally, there have been introduce two boundaries or chromatin insulators each site of our system. They are transcriptional terminator that act as neutral transcriptionally elements that prevent negative or positive influence from extending across chromatin to a promoter (Fourel et al, 2001; Bushey et al, 2008).
[Figure 5]. Reporter module:. Kan-MX6 (selectable cassette for integration in leu1), Adh1 terminator (upstream insulator ), tetO2 (upstream operator sites), urg1 promoter, GFP marker gene, Adh1 terminator, tetO4 (downstream operator sites), Act1 terminator (downstream insulator).
Sponsors
