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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.

Figure 2. Digestion of pMA-RQ synthetic plasmid containing Tadh1-tetO2 part and pfa6a-MX6-Purg plasmid with BglII results. Analytical digestions of pfa6a-MX6-Purg plasmid

Figure 3. Screening of positives clones of Tadh1-tetO2 into pfa6a-MX6-Purg plasmid

Figure 4. Digestion results of (Tadh1·tetO2)+pfa6a-MX6-Purg plasmid and GFP·Tadh1·tetO4 synthetic construct.

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 5. 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.

Figure 6. Digestion of synthetic plasmid containing engineered silencing protein tetR-CSD and pREP41X plasmid with XhoI and XmaI restriction enzymes. Swi6 gene, amplified by PCR reaction.

Figure 7. Analytical digestions of the positive clones of pREP41X plasmid plus the engineered silencing protein, tetRCSD, with XhoI and XmaI restriction enzymes.

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.