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− | We've developed three interleukin biosensors of varying complexity. Our overall biosensor design is based on the expression of interleukin receptors on the surface of yeast cells. Upon binding interleukins, two receptor constructs associate, leading to the activation of an intracellular signaling cascade. The receptor constructs consist of the extracellular part of an interleukin receptor fused to a generic transmembrane domain (TMD) (Wsc1, [1]) and to an intracellular, split actuator protein. For the three designs, these split proteins are; | + | We've developed three interleukin biosensors of varying complexity. Our overall biosensor design is based on the expression of interleukin receptors on the surface of yeast cells. Upon binding interleukins, two receptor constructs associate, leading to the activation of an intracellular signaling cascade. The receptor constructs consist of the extracellular part of an interleukin receptor fused to a generic transmembrane domain (TMD) (Wsc1 [1]) and to an intracellular, split actuator protein. For the three designs, these split proteins are; |
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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) [1], 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 [2]). 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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− | 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 [3]. 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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| To further improve the potential sensing properties of our biosensor we decided to exploit signal processing capabilities of the yeast pheromone sensing pathway by integrating parts of it into our biosensor design. This was anticipated to result in higher biosensor sensitivity.<br><br> | | To further improve the potential sensing properties of our biosensor we decided to exploit signal processing capabilities of the yeast pheromone sensing pathway by integrating parts of it into our biosensor design. This was anticipated to result in higher biosensor sensitivity.<br><br> |
− | Yeast has a G-protein-coupled receptor (GPCR) specific to yeast mating pheromones. When a pheromone binds to the receptor, the receptor occupancy stimulates the G-alpha subunit of the G-protein to exchange GDP for GTP, and release the beta and gamma subunits. The released beta and gamma subunits can then recruit the Ste5 scaffold protein to the membrane, starting a phosphorylation cascade eventually leading to the phosphorylation and activation of the transcription factor Ste12 and expression of pheromone response genes [5]. | + | Yeast has a G-protein-coupled receptor (GPCR) specific to yeast mating pheromones. When a pheromone binds to the receptor, the receptor occupancy stimulates the G-alpha subunit of the G-protein to exchange GDP for GTP, and release the beta and gamma subunits. The released beta and gamma subunits can then recruit the Ste5 scaffold protein to the membrane, starting a phosphorylation cascade eventually leading to the phosphorylation and activation of the transcription factor Ste12 and expression of pheromone response genes [4]. |
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| Since the dissociation of G-alpha from the beta and gamma subunits is what drives the pheromone cascade, we decided to design our own switch for this step in the pathway. Building onto the previous TEV protease design, we designed a mutant G-alpha with cleavage sites from the TEV protease inserted at multiple points, as described on our engineering success page. This meant that the presence of an interleukin, and ultimately the reconstitution of the TEV protease, would result in the cleavage of G-alpha into multiple fragments. These fragments would then, in theory, dissociate from the beta/gamma subunit, freeing it in a similar fashion to the normal signal transduction pathway. The free beta/gamma subunit would then be able to trigger the pheromone pathway and activate a downstream reporter gene. In order to maintain orthogonality, we used the same promoter as previously, and the transcription factor LexA-Ste12 - a synthetic transcription factor that can be activated by phosphorylation in the last step of the phosphorylation cascade in the pheromone pathway. | | Since the dissociation of G-alpha from the beta and gamma subunits is what drives the pheromone cascade, we decided to design our own switch for this step in the pathway. Building onto the previous TEV protease design, we designed a mutant G-alpha with cleavage sites from the TEV protease inserted at multiple points, as described on our engineering success page. This meant that the presence of an interleukin, and ultimately the reconstitution of the TEV protease, would result in the cleavage of G-alpha into multiple fragments. These fragments would then, in theory, dissociate from the beta/gamma subunit, freeing it in a similar fashion to the normal signal transduction pathway. The free beta/gamma subunit would then be able to trigger the pheromone pathway and activate a downstream reporter gene. In order to maintain orthogonality, we used the same promoter as previously, and the transcription factor LexA-Ste12 - a synthetic transcription factor that can be activated by phosphorylation in the last step of the phosphorylation cascade in the pheromone pathway. |
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| <div class="title">Attributions</div> | | <div class="title">Attributions</div> |
− | <div class="text">If not already cited in other sections of your poster, what literature sources did you reference on this poster? Who helped or advised you? </div> | + | <div class="text"> |
| + | <b>Supervisors:</b> Sotirios Kampranis, Nanna Heinz, Karel Miettinen, Jon Fugl, Nattawat Leelahakorn, Cecilie Cetti Hansen, Iben Egebæk Nikolajsen & Jonas Hansen. |
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| + | <b>Thank you to our sponsors!</b> |
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| + | <b>Scientific references</b> |
| + | [1] - Snider, J., Kittanakom, S., Curak, J., & Stagljar, I. (2010). Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: A powerful tool for identifying protein-protein interactions. Journal of Visualized Experiments. https://doi.org/10.3791/1698↩ |
| + | [2] - Dossani, Zain & Apel, Amanda & Szmidt‐Middleton, Heather & Hillson, Nathan & Deutch, Samuel & Keasling, Jay & Mukhopadhyay, Aindrila. (2017). A combinatorial approach to Synthetic Transcription Factor-Promoter combinations for yeast strain engineering. Yeast. 35. 10.1002/yea.3292. |
| + | [3] - Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., Bach, A., Nave, K. A., & Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods. https://doi.org/10.1038/nmeth967 |
| + | [4] - Bardwell L. A walk-through of the yeast mating pheromone response pathway. Peptides. 2005;26(2):339-350. doi:10.1016/j.peptides.2004.10.002 |
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