Part:BBa_K1391112:Experience

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Applications of BBa_K1391112

Cofilin is an actin binding protein that has been shown to also act as a cofactor when interacting with the receptors LilrB2 and PirB. When these receptors are activated, they recruit cofilin, which then gets activated itself. Inactive cofilin is phosphorylated at the third amino acid and it gets activated by de-phosphorylation by the receptors. The sequence for this part encodes an amino acid at the third position that is different from naturally occurring cofilin. This provides a "pseudophosphrylated" effect that we hope to limit cross reactions between other components of the cell, increasing the orthogonality of our system.

In this detection system, LilrB2 was fused to a linker, a TEV protease (TEVp) cleavage site and a transcription factor (in that order) at its intracellular domain. Cofilin was fused to TEV protease. These modifications allowed the manipulation of the natural operational system of LilrB2 such that when beta-amyloid oligomers bind to the receptor (and activate it) the TEV protease on the recruited cofilin cleaves at the TEVp cleavage site. This releases the transcription factor in to the cytosol, where it is guided to the nucleus of the cell and activates some subsequent (reporter or treatment) module.

In this part, expression of cofilin can be controlled by the amount of doxycycline introduced to the cell medium via a tetracycline responsive promoter.

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