Team:UPO-Sevilla/Project/Epigenetic Flip Flop/Designing
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The epigenetic bistable has two main modules: reporter module and compaction module.
Reporter Module contains a GFP inducible expression system, flanked by tetR 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; Bähler et al, 1998; Watt et al, 2008).
Figure 1. 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).
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 2. 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.
Bioinformatics notes: Key proteins
1. TetR (Uniprot: D8E9H0)
Table 1. Relevant information of TetR protein from Uniprot protein database entry D8E9H0 and TetR Pfam database.
2. Swi6 (Uniprot: P40381)
3. Sir3 (Uniprot: P0670)
Figure 5. Sir3 BAM domain structure predicted by Swiss-Model homology modelling server.