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| − | <p><strong><span data-contrast="auto">Remember to write about the background first (</span></strong><strong><span data-contrast="auto">ie</span></strong><strong><span data-contrast="auto"> that there even is a conc in sweat that correlates to the amount in blood)</span></strong><strong><span data-contrast="auto"> wait this should probably be under the project description? I think?</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">IL-1</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">As mentioned above, the mechanism of action of IL-1 binding and signaling relies on the association of two or more receptors</span><span data-contrast="auto"> and the interleukin itself</span><span data-contrast="auto">.</span><span data-contrast="auto"> The receptors in question are the IL-1R and the accessory receptor IL1RAcP. Formation of the heterotrimer and binding of the interleukin results in activation of the pathway in the native setting.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Our first step when working with IL-1 was to </span><span data-contrast="auto">look into</span><span data-contrast="auto"> the IL-1R f</span><span data-contrast="auto">amily. Through our search, we found that there are some receptors that function as inhibitors of the IL-1 system, and that could be </span><span data-contrast="auto">more </span><span data-contrast="auto">useful </span><span data-contrast="auto">in </span><span data-contrast="auto">our context</span><span data-contrast="auto"> compared to the signaling IL-1RI receptor</span><span data-contrast="auto">. </span><span data-contrast="auto">Particularly</span><span data-contrast="auto">,</span><span data-contrast="auto"> </span><span data-contrast="auto">the IL-1RII decoy recept</span><span data-contrast="auto">or was of interest to us. The IL-1RII receptor differs from the type 1 receptor in that it lacks an intracellular toll-like receptor essential for </span><span data-contrast="auto">normal </span><span data-contrast="auto">signal relay,</span><span data-contrast="auto"> but </span><span data-contrast="auto">it</span><span data-contrast="auto"> has some </span><span data-contrast="auto">nice features</span><span data-contrast="auto"> of interest to us</span><span data-contrast="auto">. </span><span data-contrast="auto">For example, it has lower affinity for the IL-1 antagonist compared to the type 1 receptor</span><span data-contrast="auto"> (SOURCE)</span><span data-contrast="auto">, which is </span><span data-contrast="auto">great for us, as it increases the selectivity for the signaling IL-1 molecules.</span><span data-contrast="auto"> It’s because of this increased sensitivity, and because the intracellular signaling domains are of no importance in our context, we decided to use the IL-1RII extracellularly.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">IL-6</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
| |
| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
| |
| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
| |
| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">IL-10</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
| |
| − | <p><span data-contrast="auto">The receptors for IL-10 form heterotrimers like the receptors for IL-1. Here, the IL-10R type 1 associated with IL-10, and subsequently recruits IL-10R type II. The reason we decided to </span><span data-contrast="auto">look into</span><span data-contrast="auto"> IL-10 is to have a </span><span data-contrast="auto">non</span><span data-contrast="auto"> acute</span><span data-contrast="auto">-phase cytokine in our system. This receptor is also much smaller than the previous two receptors, </span><span data-contrast="auto">increasing the chance of correct folding in S. cerevisiae, and is located closer to the membrane compared to the IL-6 receptor for instance.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">Very good article: </span><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489423/"><span data-contrast="none"><span data-ccp-charstyle="Hyperlink">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489423/</span></span></a><span data-contrast="none"> Source????? Is this it???</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">Intro middle text:</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
| |
| − | <p><span data-contrast="auto">The extracellular portions of the human interleukin receptors were then fused to a transmembrane domain to secure localization to the membrane. For this, we</span><span data-contrast="auto"> </span><span data-contrast="auto">researched</span><span data-contrast="auto"> a lot of different endogenous yeast transmembrane domains (hereafter called TMDs)</span><span data-contrast="auto"> to find the one with the highest </span><span data-contrast="auto">predicted </span><span data-contrast="auto">localization to the membrane</span><span data-contrast="auto">, based on sequence analyses. We ran a sequence </span><span data-contrast="auto">analysis</span><span data-contrast="auto"> on 13 different endogenous single-pass </span><span data-contrast="auto">type I </span><span data-contrast="auto">transmembrane proteins as part of this </span><span data-contrast="auto">endeavor, and</span><span data-contrast="auto"> using our knowledge of the characteristics of the phospholipid bilayer and transmembrane proteins in general, we came up with our own </span><span data-contrast="auto">candidate for a transmembrane protein. This TMD would have hydrophobic amino acid residues pointing “inwards”, with the polar amino acid residues on either side, and tryptophan in</span><span data-contrast="auto">-between those areas. Our candidate had the following sequence of amino acids:</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">IAGIVIGVVLGVIFILIAILFAFW</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">And proved to have a higher predicted localization to the membrane than the TMDs we compared it to (SOURCE? PIC? DRY LAB?). </span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">As this added a level of unpredictability though, we decided to use the TMD</span><span data-contrast="auto"> Wsc1 for our project design henceforth, as some of our designs build on papers that use the Wsc1 domain.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">The next step was to imitate an intracellular transduction pathway, following the extracellular association of our receptor complexes. As all our interleukin receptors’ normal mechanisms of action build on two non-identical receptors associating, we thought to integrate a similar protein/protein interaction as the key part of our own transduction system.</span><span data-contrast="auto"> This gave rise to our first design:</span><span data-contrast="auto"> the split-ubiquitin design.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">Ubiquitin</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">This was the first design we developed, after being introduced to t</span><span data-contrast="auto">ools</span><span data-contrast="auto"> </span><span data-contrast="auto">for identifying protein-protein interactions by our supervisor. Here we found the split-ubiquitin </span><span data-contrast="auto">based Membrane Yeast Two-Hybrid </span><span data-contrast="auto">method</span><span data-contrast="auto"> (MYTH) (SOURCE: </span><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818708/"><span data-contrast="none"><span data-ccp-charstyle="Hyperlink">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818708/</span></span></a><span data-contrast="auto"> )</span><span data-contrast="auto">, where </span><span data-contrast="auto">two different </span><span data-contrast="auto">protein</span><span data-contrast="auto">s of interest</span><span data-contrast="auto"> </span><span data-contrast="auto">are</span><span data-contrast="auto"> fused to one half of a modified ubiquitin molecule</span><span data-contrast="auto"> each</span><span data-contrast="auto">. </span><span data-contrast="auto">The modifications made to the </span><span data-contrast="auto">two halves of </span><span data-contrast="auto">u</span><span data-contrast="auto">biquitin renders </span><span data-contrast="auto">them</span><span data-contrast="auto"> unable to spontaneously bind to each other and reconstitute,</span><span data-contrast="auto"> without </span><span data-contrast="auto">first </span><span data-contrast="auto">being brought into </span><span data-contrast="auto">close proximity</span><span data-contrast="auto"> of each other by another protein</span><span data-contrast="auto">. This means that if the proteins of interest, that the ubiquitin halves are fused to, have a natural affinity for each other and consequently associate, the ubiquitin halves will also associate on the intracellular side of the membrane and reconstitute, as they’re being brought closer together by the </span><span data-contrast="auto">association on the extracellular side</span><span data-contrast="auto">.</span><span data-contrast="auto"> This suits our purposes perfectly, as we</span><span data-contrast="auto"> already know that the interleukin receptor parts that we’re using have a natural affinity for each other in the native setting</span><span data-contrast="auto"> (and of course in the presence of the fitting interleukin)</span><span data-contrast="auto">.</span><span data-contrast="auto"> Thus, by fusing ubiquitin to each of our receptors, we know that an extracellular association will result in an intracellular reconstitution of ubiquitin.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">BOX:</span><span data-contrast="auto"> Modifying the ubiquitin halves happens through switching amino acid number 13 out from </span><span data-contrast="auto">isoleucin</span><span data-contrast="auto"> to glycine.</span><span data-contrast="auto"> The C-terminal half (Cub) will then contain 42 aa, while the N-terminal half (Nub) will have a length of 51 aa</span><span data-contrast="auto">, following the MYTH system proposed by Snider, J. et al. 2010.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">At the same time as we fuse the two halves of ubiquitin to our two receptor proteins, we fuse a transcription factor to the C-terminal part of ubiquitin. </span><span data-contrast="auto">As ubiquitin is recognized by deubiquitinating enzymes upon reconstitution, this means that the deubiquitinating enzymes will cleave </span><span data-contrast="auto">off the transcription factor bound to ubiquitin, resulting in the release of the free transcription factor into the cytosol and ultimately the cell nucleus, where it can exert it</span><span data-contrast="auto">s effect.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">For our purposes, we’ve using a synthetic transcription factor developed by </span><span data-contrast="auto">Dossani, Z. Y. et al. 2018. This consists of the bacterial </span><span data-contrast="auto">LexA</span><span data-contrast="auto"> DNA binding protein, fused with the viral activator domain </span><span data-contrast="auto">VP16</span><span data-contrast="auto">. A corresponding hybrid promoter with </span><span data-contrast="auto">operator regions replaced with sequences that are recognized by </span><span data-contrast="auto">LexA</span><span data-contrast="auto"> is also used, to avoid transcription of yeast-native genes</span><span data-contrast="auto"> </span><span data-contrast="auto">(SOURCE:</span><span data-contrast="auto"> </span><a href="https://pubmed.ncbi.nlm.nih.gov/29084380/"><span data-contrast="none"><span data-ccp-charstyle="Hyperlink">https://pubmed.ncbi.nlm.nih.gov/29084380/</span></span></a><span data-contrast="auto"> )</span><span data-contrast="auto">.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">INSERT</span><span data-contrast="auto">: picture of ubiquitin design</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">However, as mentioned in the project description page, the concentrations of interleukins found in sweat are rather low, and as of now, this design has no</span><span data-contrast="auto"> real</span><span data-contrast="auto"> signal amplific</span><span data-contrast="auto">ation step. On the </span><span data-contrast="auto">other hand</span><span data-contrast="auto">,</span><span data-contrast="auto"> the </span><span data-contrast="auto">close resemblance to the </span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Under normal circumstances we would’ve tested our design in the wet lab to gauge the importance of amplification at this point, </span><span data-contrast="auto">or waited for the dry lab results</span><span data-contrast="auto"> to see if they confirmed our suspicions</span><span data-contrast="auto">, </span><span data-contrast="auto">but</span><span data-contrast="auto"> due to the restrictions in the lab we</span><span data-contrast="auto"> </span><span data-contrast="auto">instead decided to re-think our </span><span data-contrast="auto">design from the get-go. This meant that the next step for us would be to make another design, now with preferably more amplification.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">INSERT: </span><span data-contrast="auto">engineering cycle figure</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">Transcription</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Our i</span><span data-contrast="auto">ntermediate </span><span data-contrast="auto">design utilizes the same receptor-system as the ubiquitin-based design, but with few modifications. Thanks to our supervisor</span><span data-contrast="auto">’s guidance, we were introduced to another kind of split-protein: the split TEV-protease.</span><span data-contrast="auto"> This was another method developed by Wehr, M. </span><span data-contrast="auto">C. </span><span data-contrast="auto">et al. In 2006 to monitor protein-protein interactions. Here, we again have two engineered inactive halves of the TEV-protease, that only regain activity</span><span data-contrast="auto"> when </span><span data-contrast="auto">coexpressed</span><span data-contrast="auto"> as fusion constructs with interacting proteins (SOURCE: </span><a href="https://www.nature.com/articles/nmeth967"><span data-contrast="none"><span data-ccp-charstyle="Hyperlink">https://www.nature.com/articles/nmeth967</span></span></a><span data-contrast="auto"> ). Therefore, we again utilize the receptor/TMD domains from the previous designs, but now each of our receptors will be fused to one half of the TEV-protease instead.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">BLAHBLAHBLAH write about the linker between the protease and the other shit</span><span data-contrast="none"> with how you make it blah blah. From benchling:</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">The TEV N1a protease design is to have N-TEV with the aa residues 1-118 from the TEV protein, and a C-TEV from 119-242, as laid out by the original designers of the protease (Wehr, M. C. 2006), which will then be fused to the C-terminal ends of the transmembrane proteins. The N-TEV can also be fused to something else on the N-terminal without loss of function. According to a later article by the same author though (</span><span data-contrast="none">Wintgen</span><span data-contrast="none">, J. P. et al. 2017) there's also a substitution at residue 219, where a Serine is changed out for a Proline (Ser --> Pro, also covered by Tropea, J. E. 2009). Additionally, an optimization can be made where the protease is truncated after amino acid 221 to remove the inhibitory C-terminal tail, which blocks the active site of the TEV protease, and TEVs up to 231 aa are sold on </span><span data-contrast="none">genscript</span><span data-contrast="none">.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">In parallel, we also express the Wsc1 TMD, which will be sorted and localized to the membrane. To this TMD, we’ll fuse the same transcription factor from the previous design (LexA-VP16</span><span data-contrast="auto">), and</span><span data-contrast="auto"> use the recognition sequence for the TEV-</span><span data-contrast="auto">protease as the linker between the two.</span><span data-contrast="auto"> This means that the TEV-protease, upon reconstitution, will be able to cleave the transcription factor and free it into the cytosol. In theory, the TEV-protease will be able to cut many transcription factors loose, meaning that one interleukin (</span><span data-contrast="auto">by extension of</span><span data-contrast="auto"> the association of our two receptors)</span><span data-contrast="auto"> will result in the cleavage of multiple transcription factors and thus an amplification of the signal.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Again, this extra amplification step made us hopeful, but in order to achieve the highest level of amplification possible we moved on to other venues.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><strong><span data-contrast="auto">G alpha</span></strong><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Our </span><span data-contrast="auto">last and </span><span data-contrast="auto">most ambitious design</span><span data-contrast="auto"> hinges on hijacking the pheromone pathway in yeast.</span><span data-contrast="auto"> We had no doubt that this would give us the most amplification</span><span data-contrast="auto">, which is especially important in our case given the low concentrations of interleukins in sweat (SOURCE</span><span data-contrast="auto">????). The pheromone pathway is XXXXX EXPLAIN HERE OR NO?</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">In order to hijack the pheromone pathway, we initially set out to understand how the Ste5 scaffold protein was recruited to the membrane by the beta/gamma complex. Soon though, we saw that the interaction between the beta/gamma complex and the scaffold was </span><span data-contrast="auto">more complicated than previously </span><span data-contrast="auto">thought, and</span><span data-contrast="auto"> introducing changes to this step seemed </span><span data-contrast="auto">risky </span><span data-contrast="auto">(SOURCE about how we don’t know what beta/gamma does exactly here). Since the exact mechanism by</span><span data-contrast="auto"> which the beta/gamma complex recruits Ste5 was unknown to us, we decided to take a more conservative approach</span><span data-contrast="auto"> instead of </span><span data-contrast="auto">changing beta/gamma for another recruiting </span><span data-contrast="auto">maneuvre</span><span data-contrast="auto">. Here, we finally thought to introduce an inhibitory sequence on beta, </span><span data-contrast="auto">that could be removed at will, </span><span data-contrast="auto">so as to</span><span data-contrast="auto"> keep the beta/gamma complex as the recruiting element</span><span data-contrast="auto">. This inhibitory sequence would then, building onto the previous design, contain the TEV protease cleavage site, so</span><span data-contrast="auto"> an extracellular signal could trigger the TEV protease to cut off the inhibitory sequence and start the signaling. The next step from here would be to design such an inhibitory sequence, and through talking to</span><span data-contrast="auto"> our supervisor, we agreed that we should stay in the same conservative vein and look at natural inhibitors of the signaling. Here, the alpha subunit of the G protein was an obvious choice, as the alpha subunit binds to beta/gamma in the absence of an extracellular </span><span data-contrast="auto">signal, and</span><span data-contrast="auto"> hinders them from recruiting Ste5 and completing the pheromone pathway signal</span><span data-contrast="auto">ing. As a natural inhibitor, G alpha was a great choice for us.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">The next step from here was to find places to insert the TEV protease cleavage sites in G alpha. Our goals when doing this was to have a G alpha that</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">1) could keep its GTPase activity, so it could be used in other contexts in the future</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">2) could bind to and exert an inhibitory function on the beta/gamma complex’s ability to recruit Ste5</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">3) could, once cut by the TEV protease in the presence of a signal, dissociate from beta/gamma again, enabling regular signaling and recruitment of Ste5</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">For this, we had many approaches. Our first thought was to look at the yeast G alpha’s </span><span data-contrast="auto">sequence, and</span><span data-contrast="auto"> change very few amino acids in places that already resembled the TEV recognition</span><span data-contrast="auto"> site. During this first iteration, we used the following recognition site:</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic and φ is any small hydrophobic or polar residue (S/</span><span data-contrast="none">G;</span><span data-contrast="none"> </span><a href="https://pubmed.ncbi.nlm.nih.gov/15477088/"><span data-contrast="none"><span data-ccp-charstyle="Hyperlink">https://pubmed.ncbi.nlm.nih.gov/15477088/</span></span></a><span data-contrast="none">).</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">(INSERT PRELIMINARY RESULTS?)</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">However, we found that introducing different amino acids (albeit with the same properties) in the different cleavage sites would prove to</span><span data-contrast="none"> make subsequent wet lab troubleshooting harder, as well as change the dry lab output in unr</span><span data-contrast="none">easonable </span><span data-contrast="none">ways</span><span data-contrast="none">, as the TEV protease would then have different efficiencies depending on the used recognition site</span><span data-contrast="none"> (INSERT SOURCE DAVID’S ARTICLE FROM BENCHLING)</span><span data-contrast="none">.</span><span data-contrast="none"> In order to limit the amount of unknowns</span><span data-contrast="none">, and to have the same affinity of our TEV protease to our recognition site, we opted to use the same recognition site everywhere</span><span data-contrast="none">; ENLYFQG.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">Using mutagenesis results from </span><span data-contrast="none">uniprot</span><span data-contrast="none"> and other literature (SOURCE</span><span data-contrast="none">???? May be weird, I’m sleepy</span><span data-contrast="none">) we found the residues that have be</span><span data-contrast="none">e</span><span data-contrast="none">n found to </span><span data-contrast="none">give </span><span data-contrast="none">constitutive activity in the pheromone pathway as a result of alpha not being able to bind to beta/gamma (SOURCE inser</span><span data-contrast="none">t </span><span data-contrast="none">Gladue</span><span data-contrast="none">, D. P. 2008)</span><span data-contrast="none"> etc</span><span data-contrast="none">etera, and through extensive computer modeling described under our dry lab section, we eventually landed on some G alpha mutants where the TEV recognition site is inserted into tactical, most promising areas that should not interfere with normal G alpha function.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">So, to summarize, our last and final design entails:</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">1) A signaling interleukin reaches our two receptors</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">2) The receptors associate extracellularly, giving rise to the intracellular complementation of a split TEV protease</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">3) The now active TEV protease can cut a mutant G alpha protein</span><span data-contrast="none"> (LINK TO ENGINEERING SUCCESS PAGE!!!)</span><span data-contrast="none"> into smaller peptide fragments, that’ll dissociate from the beta/gamma complex</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">4) The beta/gamma complex can exert its recruiting function on the Ste5 scaffold protein and recruit it to the membrane, thus triggering the pheromone cascade</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">5) In the end, our modified transcription factor, LexA-VP16, will bind to </span><span data-contrast="none">X</span><span data-contrast="none"> promoter and result in the transcription and translation of our reporter protein</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">This last and final design has a lot of strengths compared to the two prior designs, but also some </span><span data-contrast="none">drawbacks....</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="none">HOW DO WE SAY THAT WE USED THE ENGINEERING CYCLE PERFECTLY, AND WHERE DO I PUT MY FIGURE???????</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">----- (notes)</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Remember to say that our idea was to put an inhibitory sequence on the beta because we don’t know what beta/gamma does to recruit the Ste5 protein thing (source!!!), and then thanks to Karel we know something now.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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| − | <p><span data-contrast="auto">Remember to say that in order to limit the amount of unknowns (different affinity sites and cleavage) we used the same recognition site.</span><span data-ccp-props="{"201341983":0,"335559739":160,"335559740":259}"> </span></p>
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