Team:UPO-Sevilla/Project/Epigenetic Flip Flop/Procedure and Results

From 2011.igem.org

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                             <h1>Procedure and Results</h1>
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<p>In order to construct the two modules of the epigenetic toogle switch, we have used tools like the plasmids pF6a-MX6-Kan-Purg-GFP and pREP41X already available. These plasmids will be modified to introduce into them the epigenetic control elements. For further information, please read <a href="https://2011.igem.org/Team:UPO-Sevilla/Project/Notebook/Epigenetic_Flip_Flop" target="_blank" title="Epigenetic Flip Flop Notebook">Epigenetic Flip Flop Notebook</a>.</p>
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<p>In order to construct the two modules of the epigenetic toggle switch, we have used tools like the plasmids pF6a-MX6-Kan-Purg-GFP and pREP41X already available. These plasmids will be modified to introduce into them the epigenetic control elements. For further information, please read <a href="https://2011.igem.org/Team:UPO-Sevilla/Project/Notebook/Epigenetic_Flip_Flop" target="_blank" title="Epigenetic Flip Flop Notebook">Epigenetic Flip Flop Notebook</a>.</p>
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     <div class="centre"><img src="https://static.igem.org/mediawiki/2011/6/65/UPOSevilla_AssemblyReporterModule.png" width="700px" height="500px"></div>
     <div class="centre"><img src="https://static.igem.org/mediawiki/2011/6/65/UPOSevilla_AssemblyReporterModule.png" width="700px" height="500px"></div>
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<p><strong>Achievements</strong></p>
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<p>- Construction of the reporter module and its corresponding controls, as explained. </p>
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<p>- Stochastic integration of the reporter module in S. pombe genome to study the effect of heterochromatin context in the functioning epigenetic toggle switch.</p>
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<p>- Amplification of the reporter module with special primers and integration into leu1 locus of S. pombe by homologous recombination.</p>
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<p><strong>Achievements</strong></p>
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<p>By the wiki freeze: </p>
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<p>- Cloning of tetR-CSD into pREP41X plasmid. Transformation into the strain generated that contains the reporter module, and its controls.</p>
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<p>- The reporter module has already been constructed. This construction will be amplified with special primers and integrated into leu1 locus of <em>S. pombe</em> by homologous recombination, and also it will be stochastically integrated in its genome.</p>
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<p>- Amplification of Sir3 and Swi6 open reading frames  by PCR.</p>
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<p>- The  compaction module tetR-CSD is finally cloned into the pREP41X plasmid, and it will be transformed into the strain generated that contains the reporter module. The Sir3 and Swi6 fragments have been obtained by PCR and we are now cloning them into the pREP41X plasmid, fussed to tetR. </p>
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Revision as of 12:15, 21 October 2011

Grey iGEM Logo UPO icon

Procedure and Results

In order to construct the two modules of the epigenetic toggle switch, we have used tools like the plasmids pF6a-MX6-Kan-Purg-GFP and pREP41X already available. These plasmids will be modified to introduce into them the epigenetic control elements. For further information, please read Epigenetic Flip Flop Notebook.


[Figure 1]. The assembly process of the reporter module involved two cloning steps: first, insertion of adh1 transcriptional terminator plus two tetracycline repeats (Tadh1-tetO2) upstream of urg1 promoter, using the BglII restriction site, and second, insertion of the reporter gene plus T adh1 plus four tetracycline repeats plus actin transcriptional terminator (GFP-Tadh1-tetO4-Tact1) downstream of urg1 promoter, using PacI/AscI sites. The composite parts were obtained by a DNA synthesis.


P urg1 tetO

Purg1 + tetO screening

GFP-tetO4



Achievements

- Construction of the reporter module and its corresponding controls, as explained.

- Stochastic integration of the reporter module in S. pombe genome to study the effect of heterochromatin context in the functioning epigenetic toggle switch.

- Amplification of the reporter module with special primers and integration into leu1 locus of S. pombe by homologous recombination.


[Figure 2]. The assembly process of compaction module involved a single cloning step. The three optional engineered silencing proteins will be cloned in the polylinker of pREP41X (nmt41 promoter and nmt1 terminator), using the restriction sites XhoI and XmaI/SmaI. The insert containing tetracycline repressor plus chromoshadow domain of Swi6 (TetR-CSD) was obtained by DNA synthesis, and the inserts containing tetracycline repressor fussed either to sir3 or swi6 were amplified by PCR in the laboratory using pPR013 (Dr Attila Becskei Lab, Univertity of Zurich) and genomic DNA of S. pombe as templates.


Compaction module assembly gel

Achievements

- Cloning of tetR-CSD into pREP41X plasmid. Transformation into the strain generated that contains the reporter module, and its controls.

- Amplification of Sir3 and Swi6 open reading frames by PCR.