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| The initial, minimal, biosensor design builds on the split-ubiquitin based Membrane Yeast Two-Hybrid method (MYTH) [2], where two proteins of interest are fused to either half of a modified ubiquitin molecule, that are by themselves unable to reconstitute a functional protein. However, whenever the proteins of interest associate, the attached halves of split ubiquitin come into proximity of each other, facilitating reconstitution. In addition, one of the halves of split ubiquitin is further fused to a synthetic transcription factor (LexA-VP16 [3]). Upon reconstitution, the ubiquitin can be cleaved by deubiquitinating enzymes resulting in the release of the transcription factor and ultimately the activation of a reporter gene resulting in a biosensor signal. | | The initial, minimal, biosensor design builds on the split-ubiquitin based Membrane Yeast Two-Hybrid method (MYTH) [2], where two proteins of interest are fused to either half of a modified ubiquitin molecule, that are by themselves unable to reconstitute a functional protein. However, whenever the proteins of interest associate, the attached halves of split ubiquitin come into proximity of each other, facilitating reconstitution. In addition, one of the halves of split ubiquitin is further fused to a synthetic transcription factor (LexA-VP16 [3]). Upon reconstitution, the ubiquitin can be cleaved by deubiquitinating enzymes resulting in the release of the transcription factor and ultimately the activation of a reporter gene resulting in a biosensor signal. |
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| + | <img src=""> |
| + | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;"><b>Fig. 2. Minimal biosensor design. </b> |
| + | Complementation of the C- and N-terminal halves of Ubiquitin results in their recognition by deubiquitinating enzymes, leading to cleavage and release of the bound LexA-VP16 transcription factor.</figcaption> |
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| In order to add one amplification step to our biosensors signaling pathway we devised another biosensor design. Our intermediate design utilizes a similar receptor-system to the ubiquitin-based design. Here, our intracellular split-protein is the split TEV-protease. This is another method to monitor protein-protein interactions, described by Wehr, M. C. et al in 2006. Here, we again have two engineered inactive halves of the TEV-protease, that only regain activity when coexpressed as fusion constructs with interacting proteins [4]. Therefore, we again utilize the receptor/TMD from the previous design, but now each of our receptors will be fused to one half of the TEV-protease instead with a flexible linker.<br><br> | | In order to add one amplification step to our biosensors signaling pathway we devised another biosensor design. Our intermediate design utilizes a similar receptor-system to the ubiquitin-based design. Here, our intracellular split-protein is the split TEV-protease. This is another method to monitor protein-protein interactions, described by Wehr, M. C. et al in 2006. Here, we again have two engineered inactive halves of the TEV-protease, that only regain activity when coexpressed as fusion constructs with interacting proteins [4]. Therefore, we again utilize the receptor/TMD from the previous design, but now each of our receptors will be fused to one half of the TEV-protease instead with a flexible linker.<br><br> |
| In parallel, we also express the Wsc1 TMD, which is fused with the same transcription factor from the previous design, and use the recognition sequence for the TEV-protease as the linker between the two. Thus, upon reconstitution,The TEV protease will be able to cleave the transcription factor that can now freely translocate to the nucleus and activate a reporter gene. In theory, the TEV-protease will be able to cut many transcription factors loose, meaning that one interleukin (by extension of the association of our two receptors) will result in the cleavage of multiple transcription factors and thus an amplification of the biosensor signal. | | In parallel, we also express the Wsc1 TMD, which is fused with the same transcription factor from the previous design, and use the recognition sequence for the TEV-protease as the linker between the two. Thus, upon reconstitution,The TEV protease will be able to cleave the transcription factor that can now freely translocate to the nucleus and activate a reporter gene. In theory, the TEV-protease will be able to cut many transcription factors loose, meaning that one interleukin (by extension of the association of our two receptors) will result in the cleavage of multiple transcription factors and thus an amplification of the biosensor signal. |
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| + | <img src="https://2020.igem.org/File:T--UCopenhagen--Poster_int.png"> |
| + | <figcaption style="font-family: Avenir, Arial, Helvetica, sans-serif; font-size: 12px; line-height: 1.25;"><b>Fig. 3. Intermediate biosensor design. </b> |
| + | Split TEV protease complementation leading to cleavage of the linker tethering the transcription factor LexA-VP16 to the membrane.</figcaption> |
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