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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.

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 of the epigenetic toggle switch.

[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.

Achievements

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

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