Difference between revisions of "Team:UCopenhagen/Design"

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General Design of the Interleukin Yeast Biosensor

Figure 1: General mechanism of our biosensor.
Ligand binding to the extracellular receptor domain causes a conformational change, thereby initiating gene expression of a reporter gene.

In humans, interleukin mediated signaling is implemented by the association of two or more interleukin receptor proteins, most of which fall under the category of Receptor Tyrosine Kinases (RTKs) . Association of the receptor proteins results in their autophosphorylation, and the subsequent recruitment of multiple adaptor and effector proteins that bind to the phosphorylated sites and trigger a signaling pathway .

Yeast, however, has no such receptors. .

Therefore, in order to develop a yeast-based biosensor able to monitor biomarkers for general inflammation, such as interleukins, it is essential to develop a synthetic receptor and signal transduction system in yeast. To be able to detect the vast number of different interleukins, which are all important as inflammatory markers in different contexts, we opted to establish a flexible modular platform that will enable us to easily expand our biosensor to work with different interleukins. This is in line with, and because of, the advice of the experts we reached out to in our human practices work.

As most interleukin receptors are receptor tyrosine kinases, most interleukin receptors function based on the same principle: association. Thus, we based our system on the ability of the extracellular domains of the human interleukin receptors to associate in the presence of interleukin. We have fused these domains to transmembrane helices that, when the ligand is present, come together. This physical association of the transmembrane helices brings into proximity their intracellular parts, which are fused effector domains that transmit the signal to different reporter molecules.

The general mechanism of our biosensor is summarized in the following simple steps:

Mechanism

1. In the presence of the biomarker of interest, the extracellular receptor domains associate.
2. The association of the extracellular domains results in the intracellular complementation of a split protein.
3. Upon complementation of the split protein, the signal is transduced and arrives at the nucleus.
4. As a result, a reporter gene is expressed, translating the level of interleukins into a colorimetric output.

Venturing deeper into this page will show you how we hope to achieve this by developing our very own receptor-systems!

Choice of Interleukin Receptors

To establish our system, we initially focused on three main cytokines and their receptors, IL-1α/β, IL-6 and IL-10.

IL-1 IL-6 IL-10

IL-1

In the presence of an irritant or intruder causing inflammation, cytokines such as IL-1 are secreted by cells of the immune system in the inflamed area. When IL-1 then reaches the nearby endothelial cells, it signals to them to express certain proteins on their surface that are important for leukocyte adhesion. In this way, leukocytes in the blood are called to the site of inflammation, as they bind to the proteins the cells in this area express, before continuing their migration into the tissue . Due to the importance of this step in inflammation, and since IL-1 is so well-researched, it served as a good starting point.

As mentioned above, the mechanism of action of IL-1α/β binding and signaling relies on the association of two receptors and the interleukin itself. The receptors in question are the IL-1RI and the accessory receptor IL1RAcP, where the formation of the heterotrimer between these proteins results in the activation of the pathway in the native setting .

In humans, there are two inhibitors of IL-1 signaling: the IL-1RII decoy receptor, and the IL-1 antagonist. The IL-1RII decoy receptor differs from the type 1 receptor in that it lacks an intracellular toll-like domain essential for signal relay, and, interestingly, it has a lower affinity for the IL-1 antagonist compared to the signaling IL-1RI receptor . As we only planned to use the extracellular domains of these receptors, the IL-1RII proved to be a better choice for our application, as it’d provide us with increased selectivity for the signaling IL-1 molecules, as opposed to the antagonist of the system.

Figure 2: IL-1 receptor II association to IL-1RacP upon binding of IL-1.
IL-1 receptor binding causes the extracellular association between the IL-1 receptor accessory protein to IL-1RII.

IL-6

Interleukin-6 is one of the most researched cytokines in inflammation, and was a natural choice for us as it stimulates the production of c-reactive protein (CRP) – a common biomarker used in clinics . In humans, the mechanism for sensing IL-6 involves the heterotrimerization of the IL-6 receptor and the co-receptor glycoprotein 130 (gp130) with the IL-6 protein. Subsequently two of the IL-6:IL-6R:gp130 trimers form a dimer. This interaction causes the intracellular parts of the two gp130 proteins to transphosphorylate each other and start a signaling cascade .
We chose to try out two different designs of our extracellular IL-6 sensing modules. In one design, we link either halves of the intracellular split protein to the extracellular domains of the soluble isoform of gp130 (sgp130) via a transmembrane domain, while secreting a soluble isoform of IL-6R. In this way, the split protein will be reconstituted upon the dimerization of two heterotrimers.

Figure 3: Soluble IL-6 receptor heterotrimerization with sgp130 and IL-6 upon IL-6 binding.
Binding of IL-6 to sIL-6R leads to heterotrimerization with sgp130, with two trimers forming a dimer complex, thereby initiating the signaling cascade.

In the second design we use the association of IL-6 with one gp130 and one sIL-6R in a heterotrimer instead. Here we coupled the N-terminal half of the split protein to sIL-6R and the C-terminal half of the split protein to sgp130.

Figure 4: Alternative IL-6 signaling design from figure 3.
In this design, the N-terminal of the split protein is coupled to sIL-6R and the C-terminal is coupled to sgp130.

Glycoprotein 130 has six extracellular domains of which the three domains closest to the membrane mainly seem to function by positioning and bringing together the two gp130 proteins in the heterohexameric signaling complex. Since we do not depend on the association of the two gp130 proteins in the second design, we decided to use a truncated version of gp130 where we only use domain 1 to 3 in that design.
The sIL-6R has three domains; one Ig-like domain and two fibronectin-like type III domains. Only the two fibronectin-like domains (domain 2 and 3) seem to interact with the other proteins in the signaling complex . We chose to use a truncated version of the sIL-6R where we only use domain 2 and 3 since we thought this would be easier to express in our chassis.

IL-10

Finally, we chose to look at IL-10, which is an anti-inflammatory interleukin released by macrophages after the offending agent has been removed from the site of inflammation. Another difference between IL-10 and the previous two interleukins is that IL-10 is a dimer .
In IL-10 signaling, the IL-10R type I receptor associates with one domain of IL-10, and subsequently recruits the IL-10R type II. Since IL-10 is a dimer, each domain can recruit its own pair of type I and type II receptors .
Structurally, the receptor is much smaller than the previous two receptors, possibly increasing the chance of correct folding in S. cerevisiae.

Figure 5: IL-10R type I association to IL-10R type II upon IL-10 binding.
Through IL-10 binding to the IL-10R type I receptor, recruitment of IL-10R type II occurs, leading to activation of the signaling pathway.

Three Biosensor Designs

Click to go to the design sections!

Having selected the cytokines to be measured and the receptors used for their detection, we then embarked on establishing different designs for the implementation of the biosensor.

Minimal design

Intermediate design

Advanced design

Common Parts of Our Biosensor Designs

As previously mentioned, the three interleukin biosensor designs share a common mechanism of action; the association of two extracellular receptors, resulting in the intracellular association of two halves of a split protein, bound to the extracellular receptors via the same transmembrane domain. Then, they implement different signal processing strategies to achieve different levels of signal processing.

Transmembrane Domain

To ensure localization of the receptor protein fragments to the membrane, we used the Wsc1 endogenous yeast type 1 transmembrane domain, as this had previously been used for protein-protein interaction assays in yeast (see the ubiquitin design). For more information on how we decided on which transmembrane domain to use, see our modeling page.

Transcription Factor

For two of our designs we used the synthetic transcription factor LexA-VP16 variant described by Dossani, Z. Y. et al. 2018 .

This consists of the bacterial LexA DNA binding protein, fused with the viral activator domain VP16. A corresponding hybrid promoter with operator regions replaced with sequences that are recognized by LexA is also used, to avoid transcription of yeast-native genes and ensure orthogonality. The promoter we used consisted of six copies of LexO and the eno1 core promoter, in accordance with Rantasalo et. al 2016 .

Figure 6: Release of the synthetic transcription factor, LexA-VP16, leading to gene expression.
Through release of the synthetic transcription factor, LexA-VP16, in the minimal and intermediate design by cleavage of a flexible link (see below), gene expression is initiated through LexA-VP16 binding to the 6xLexO promoter.

Split Ubiquitin-Based Reporting

This first biosensor design builds on the split-ubiquitin based Membrane Yeast Two-Hybrid method (MYTH) , where two different proteins of interest are fused to one half of a modified ubiquitin molecule each. The modifications made to the two halves of ubiquitin render them unable to spontaneously bind to each other and reconstitute a functional protein, without first being brought together by another protein. 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 association on the extracellular side. This suits our purposes perfectly, as we already know that the interleukin receptor domains we use have a natural affinity for each other in the presence of their corresponding interleukin. Thus, by fusing ubiquitin to each of our receptors, we know that an extracellular association will result in an intracellular reconstitution of ubiquitin.

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. As ubiquitin is recognized by deubiquitinating enzymes upon reconstitution, this means that the deubiquitinating enzymes will cleave 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 its effect.

Figure 7: Minimal biosensor design.
Split ubiquitin complementation leading to release of C-ub bound LexA-VP16 to initiate gene expression.

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 only little amplification. On the other hand, this tried and tested method was a good, secure base to start working from.

Split TEV Protease and Membrane-bound Transcription Factor

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 . 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 with a flexible linker.

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), and use the recognition sequence for the TEV-protease as the linker between the two. 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 (by extension of the association of our two receptors) will result in the cleavage of multiple transcription factors and thus an amplification of the signal.

Figure 8: Intermediate biosensor design.
Split-TEV protease complementation leading to cleavage of a TEV-recognition sequence near LexA-VP16, thereby releasing LexA-VP16 to initiate gene expression

Hijacking the Pheromone Pathway

Our most ambitious design hinges on hijacking the pheromone pathway in yeast. This design has the potential to provide a high level of amplification, which is especially important given the low concentrations of interleukins in sweat .

Figure 9: The endogenous yeast pheromone mating pathway.
Upon alpha factor (pheromone/ligand) binding to the GPCR, intracellular signaling is initiated, and through activation of the Ste12 transcription factor, expression of genes related to the pheromone pathway is initiated.

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 .

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.

First, we set out to find places to insert the TEV protease cleavage sites in yeast G alpha (GPA1p). Our goals when doing this was to engineer a G alpha that

1. could bind to and exert an inhibitory function on the beta/gamma complex’s ability to trigger the signalling pathway cascade.
2. could, once cut by the TEV protease in the presence of a signal, dissociate from beta/gamma again, triggering the pathway.

Figure 10: Advanced biosensor design.
Split-TEV protease complementation leading to cleavage of a TEV protease cut site in GPA1, thereby initiating gene expression through the yeast pheromone pathway.

Our approach can be seen in more detail under the modeling and engineering success pages.

To summarize, our last and final design entails:

1. A signaling interleukin reaches our two receptors.
2. The receptors associate extracellularly, giving rise to the intracellular complementation of a split TEV protease.
3. The now active TEV protease can cut a mutant G alpha protein into smaller peptide fragments, that’ll dissociate from the beta/gamma complex.
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, eventually resulting in the phosphorylation of our modified LexA-Ste12 transcription factor.
5. Our activated, phosphorylated transcription factor LexA-Ste12 will bind to the synthetic promoter 6xlexo eno1, and result in the transcription and translation of our reporter protein.

Why Three Designs?

This last and final design has a lot of strengths compared to the two prior designs, but also some drawbacks. Of course, changing such a conserved protein is a great challenge, as any small change could result in great consequences for its conformation and normal functions. At the same time, however, this design proved to be the best in terms of amplification and ~~sensitivity~~, as shown through our modeling of the system compared to the two others.

For this reason, and to check whether our dry lab results fit with reality, all three of the abovementioned designs were tested in the wet lab, the results of which can be found on the results page.

Zola H. Analysis of receptors for cytokines and growth factors in human disease. Dis Markers. 1996;12(4):225-240. doi:10.1155/1996/807021 ↩
Wintheiser GA, Silberstein P. Physiology, Tyrosine Kinase Receptors. [Updated 2020 Oct 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK538532/ ↩
Gunde T, Barberis A. Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase. Biotechniques. 2005;39(4):541-549. doi:10.2144/000112011 ↩
Kaneko N, Kurata M, Yamamoto T, Morikawa S, Masumoto J. The role of interleukin-1 in general pathology. Inflamm Regen. 2019;39:12. Published 2019 Jun 6. doi:10.1186/s41232-019-0101-5 ↩
Fields James K., Günther Sebastian, Sundberg Eric J. Structural Basis of IL-1 Family Cytokine Signaling. Frontiers in Immunology. 2019;10:1412. doi:10.3389/fimmu.2019.01412↩
Garlanda, C., Riva, F., Bonavita, E., Gentile, S., & Mantovani, A. (2013). Decoys and regulatory "receptors" of the il-1/toll-like receptor superfamily. Frontiers in Immunology, 4(JUL), [Article 180]. https://doi.org/10.3389/fimmu.2013.00180 ↩
Del Giudice, Marco & Gangestad, Steven. (2018). Rethinking IL-6 and CRP: Why They Are More Than Inflammatory Biomarkers, and Why It Matters. Brain Behavior and Immunity. 70. 61-75. 10.1016/j.bbi.2018.02.013.↩
Rose-John S, Scheller J, Elson G, Jones SA. Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer. J Leukoc Biol. 2006 Aug;80(2):227-36. doi: 10.1189/jlb.1105674. Epub 2006 May 17. PMID: 16707558.↩
Kurth I, Horsten U, Pflanz S, Timmermann A, Küster A, Dahmen H, Tacken I, Heinrich PC, Müller-Newen G. Importance of the membrane-proximal extracellular domains for activation of the signal transducer glycoprotein 130. J Immunol. 2000 Jan 1;164(1):273-82. doi: 10.4049/jimmunol.164.1.273. PMID: 10605021. ↩
Zdanov A, Schalk-Hihi C, Gustchina A, Tsang M, Weatherbee J, Wlodawer A. Crystal structure of interleukin-10 reveals the functional dimer with an unexpected topological similarity to interferon gamma. Structure. 1995 Jun 15;3(6):591-601. doi: 10.1016/s0969-2126(01)00193-9. PMID: 8590020.↩
Mosser DM, Zhang X. Interleukin-10: new perspectives on an old cytokine. Immunol Rev. 2008;226:205-218. doi:10.1111/j.1600-065X.2008.00706.x↩
Rantasalo A, Czeizler E, Virtanen R, Rousu J, Lähdesmäki H, Penttilä M, et al. (2016) Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS ONE 11(2): e0148320. https://doi.org/10.1371/journal.pone.0148320↩
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↩
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↩
Hladek, Melissa & Szanton, Sarah & Cho, Young-Eun & Lai, Chen & Sacko, Caroline & Roberts, Laken & Gill, Jessica. (2017). Using sweat to measure cytokines in older adults compared to younger adults: A pilot study. Journal of Immunological Methods. 454. 10.1016/j.jim.2017.11.003. ↩
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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    <div class="everything">
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-       <h3>So... how do you want the yeast to sense interleukins?</h3>
+       <h3>General Design of the Interleukin Yeast Biosensor</h3>
-       How nice of you to ask! Yeast doesn’t have endogenous receptors for any interleukins,
+       <div class="todelt">
-      as opposed to humans. Actually, <em>Saccharomyces cerevisiae</em> doesn’t have <em>any</em>
+        <div style="display: flex; flex-direction: column; justify-content: space-between; max-width: 45%;">
-      Receptor Tyrosine Kinase type receptors (RTKs)- systems where two or more receptors associate
+          <div class="image float-left"><img src="https://static.igem.org/mediawiki/2020/8/82/T--UCopenhagen--design_system_loop.gif">
-      and start an autophosphorylation reaction, eventually leading to an intracellular signaling cascade -,
+            <figcaption><b>Figure 1: General mechanism of our biosensor.</b><br> Ligand binding to the extracellular receptor domain causes a conformational change, thereby initiating gene expression of a reporter gene. </figcaption>
-      which most interleukin receptors are <a href="#receptor" aria-describedby="footnote-label" id="receptor-ref"> </a>
+          </div>
-      <a href="#organism" aria-describedby="footnote-label" id="organism-ref"> </a>.
+        </div>
-      From the work we did in HP to understand CIDs and the patients’ everyday
+        <div class="txt-btwn"><div class="format">
-      problems and needs though, we knew we wanted to monitor biomarkers for general
+          <br>
-      inflammation, interleukins, meaning that we'd have to develop our own receptor and signal
+          <br>
-      transduction system in our yeast to sense them. At the same time, our human practices
+          In humans, interleukin mediated signaling is implemented by the association
-      work also told us about the vast number of different interleukins, all
+          of two or more interleukin receptor proteins, most of which fall under
-      important in different contexts, which influenced us to want to create a
+          the category of <b>Receptor Tyrosine Kinases (RTKs)</b> <a href="#receptor" aria-describedby="footnote-label" id="receptor-ref"> </a>. Association
-      flexible platform that enables us to easily expand the biosensor to work with different interleukins.
+          of the receptor proteins results in their autophosphorylation, and
-      As most interleukin receptors <em>are</em> receptor tyrosine kinases, though, it means
+          the subsequent recruitment of multiple adaptor and effector proteins
-      that luckily, most interleukin receptors function based on the same principle: association.
+          that bind to the phosphorylated sites and trigger a signaling
-      <br>
+          pathway <a href="#rtk" aria-describedby="footnote-label" id="rtk-ref"> </a>.
-      By using the extracellular portions of the human interleukin receptors, we
+          <br>
-      could make sure to have an association of the extracellular domains in the presence
+          <br>
-      of an interleukin, and by coupling this to the membrane through a transmembrane domain,
+          Yeast, however, has no such receptors. <a href="#no_rtk" aria-describedby="footnote-label" id="no_rtk-ref"> </a>.
-      we could couple the extracellular association of our two receptors to an
+          <br>
-      intracellular association as well. As such, the general mechanism of our biosensor would be:
+          <br>
-      <br><br>
+          Therefore, in order to develop <b>a yeast-based biosensor</b> able to monitor biomarkers
-       <li>
+          for general inflammation, such as interleukins, it is essential to develop
-	       <ol>1. In the presence of the biomarker of interest, the extracellular receptor domains will associate.</ol>
+          a synthetic receptor and signal transduction system in yeast. To be able to
-	       <ol>2. The association of the extracellular domains will result in the intracellular complementation of a split protein.</ol>
+          detect the vast number of different <b>interleukins</b>, which are all important as
-	       <ol>3. Upon complementation, the signal is transduced and arrives at the nucleus.</ol>
+          inflammatory markers in different contexts, we opted to establish a flexible
-	       <ol>4. Finally, a reporter gene is expressed, reflecting the initial level of interleukins.</ol>
+          <b>modular platform</b> that will enable us to easily expand our biosensor to work
-       </li>
+          with different interleukins. This is in line with, and because of, the advice
+          of the experts we reached out to in our <b>human practices</b> work.
+          <br><br>
+          As most interleukin receptors <em>are</em> receptor tyrosine kinases,
+          most interleukin receptors function based on the same principle: association.
+          Thus, we based our system on the ability of the extracellular domains of
+          the human interleukin receptors to associate in the presence of interleukin.
+          We have fused these domains to transmembrane helices that, when the ligand
+          is present, come together. This physical association of the transmembrane
+          helices brings into proximity their intracellular parts, which are fused
+          effector domains that transmit the signal to different reporter molecules.
+          <br>
+          <br>
+          The general mechanism of our biosensor is summarized in the following simple steps:
+          <br>
+    </div></div>
+    </div>
+    </div>
+    <div class="txt-btwn">
+    <h4 style="font-family: Montserrat; font-size: 20px;">Mechanism</h4>
+       <ol>
+	       <li><b>1.</b> In the presence of the biomarker of interest, the <b>extracellular receptor domains associate.</b></li>
+	       <li><b>2.</b> The association of the extracellular domains results in the <b>intracellular complementation</b> of a split protein.</li>
+	       <li><b>3.</b> Upon complementation of the split protein, the <b>signal</b> is transduced and arrives at the nucleus.</li>
+	       <li><b>4.</b> As a result, a reporter gene is expressed, translating the level of interleukins into a <b>colorimetric output.</b></li>
+       </ol>
        <br>
        Venturing deeper into this page will show you how we hope to achieve this by developing our very own receptor-systems!
-    </div>
+</div>
      <div class="txt-btwn">
-       <h3>Interleukin Receptors</h3>
+       <h3>Choice of Interleukin Receptors</h3>
-       This year, we’re simultaneously looking into three different interleukin
+       To establish our system, we initially focused on three main cytokines and
-       receptors, each with their own unique properties, in order to sense IL-1α/β, IL-6 and IL-10.
+       their receptors, IL-1α/β, IL-6 and IL-10.
-    </div>
+      </div>
      <!-- interleukin info boxes start here -->
@@ Line 244: / Line 320: @@
          <h3>IL-1</h3>
          <h2>IL-1</h2>
-         As mentioned above, the mechanism of action of IL-1 binding and signaling
+        In the presence of an irritant or intruder causing inflammation, cytokines
-         relies on the association of two or more receptors and the interleukin itself.
+        such as IL-1 are secreted by cells of the immune system in the inflamed
-         The receptors in question are the IL-1R and the accessory receptor IL1RAcP.
+        area. When IL-1 then reaches the nearby endothelial cells, it signals to
-         Formation of the heterotrimer and binding of the interleukin results in
+        them to express certain proteins on their surface that are important
-         activation of the pathway in the native setting. <br><br>
+        for leukocyte adhesion. In this way, leukocytes in the blood are called
+        to the site of inflammation, as they bind to the proteins the cells in
+        this area express, before continuing their migration into the tissue
+        <a href="#il1" aria-decribedby="footnote-label" id="il1-ref"> </a>. <b>Due to
+        the importance of this step in inflammation</b>, and since IL-1 is so
+        well-researched, it served as a good starting point.
+        <br>
+        <br>
+         As mentioned above, the mechanism of action of IL-1α/β binding and
+         signaling relies on the <b>association of two receptors</b> and the interleukin
+         itself. The receptors in question are the IL-1RI and the accessory receptor
+         IL1RAcP, where the formation of the heterotrimer between these proteins
+         results in the activation of the pathway in the native setting
+        <a href="#il1native" aria-decribedby="footnote-label" id="il1native-ref"> </a>.
+        <br>
+        <br>
+        In humans, there are two inhibitors of IL-1 signaling: the IL-1RII decoy
+        receptor, and the IL-1 antagonist. The IL-1RII decoy receptor differs from
+        the type 1 receptor in that it lacks an intracellular toll-like domain
+        essential for signal relay, and, interestingly, it has a lower affinity
+        for the IL-1 antagonist compared to the signaling IL-1RI receptor <a href="#antagonists" aria-describedby="footnote-label" id="antagonists-ref"> </a>.
+        As we only planned to use the extracellular domains of these receptors,
+        the IL-1RII proved to be a better choice for our application, as it’d provide
+        us with <b>increased selectivity</b> for the signaling IL-1 molecules, as opposed
+        to the antagonist of the system.<br>
-         Our first step when working with IL-1 was to look into the IL-1R family.
+         <img style="margin-left: 20%;" src="https://static.igem.org/mediawiki/2020/5/5a/T--UCopenhagen--IL1.png">
-         Through our search, we found that there are some receptors that function
+         <figcaption><b>Figure 2: IL-1 receptor II association to IL-1RacP upon binding of IL-1.</b><br> IL-1 receptor binding causes the extracellular association between the IL-1 receptor accessory protein to IL-1RII. </figcaption><br>
-        as inhibitors of the IL-1 system, and that could be more useful in our
-        context compared to the signaling IL-1RI receptor. Particularly, the IL-1RII
-        decoy receptor 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 normal signal relay, but it has some nice features of interest
-        to us. For example, it has lower affinity for the IL-1 antagonist compared
-        to the type 1 receptor (SOURCE), which is great for us, as it increases
-        the selectivity for the signaling IL-1 molecules. 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.
        </div>
@@ Line 267: / Line 357: @@
          <h3>IL-6</h3>
          <h2>IL-6</h2>
-         Interleukin-6 is one of the most researched cytokines in inflamation source. It is an acute phase mediator and generally functions pro-inflammatory in most tissues, even though it can also have anti inflammatory effects. In humans, the mechanism for sensing IL-6 involves the heterotrimerization of the IL-6 receptor and the co-receptor glykoprotein 130 (gp13) with the IL-6 protein. Subsequently two of IL-6:IL-6R:gp130 trimers form a dimer. This interaction causes the intracellular parts of the two gp130 proteins to transphosphorylate each other and start a signaling cascade.
+         Interleukin-6 is one of the most researched cytokines in inflammation, and
-<br>
+        was a natural choice for us as it stimulates the production of c-reactive
-We chose to try out two different design of our extracellular IL-6 sensing modules. In one design we link either halves of the intracellular split protein to the extracellular domains of the soluble isoform of gp130(sgp130) proteins while secreting a soluble isoform of IL-6R. Thus we utillize the association of two gp130 proteins in a heterohexamer in the first design. In the second design use the association of one gp130 and one IL-6R in a hetertrimer instead. Here we coupled the N-terminal half of the split protein to sIL-6R and the C-terminal half of the split protein to sgp130.
+        protein (CRP) – <b>a common biomarker used in clinics</b> <a href="#CRP" aria-describedby="footnote-label" id="CRP-ref"> </a>.
-<br>
+        In humans, the mechanism for sensing IL-6 involves the heterotrimerization
-Glykoprotein 130 has six extracellular domains of which the three domains closest to the membrane mainly seems to function by positioning and bringing together the two gp130 proteins in the heterohexameric signaling complex. Since we do not depend on association of the two gp130 proteins in the second design, we decided to use a truncated version of gp130 where we only use domain 1 to 3 in that design.
+        of the IL-6 receptor and the co-receptor glycoprotein 130 (gp130) with the
-<br>
+        IL-6 protein. Subsequently two of the IL-6:IL-6R:gp130 trimers form a dimer.
-The sIL-6R has three domains; one Ig-like domain and two fibronectin-like type III domains. Only the two fibronectin-like domains (domain 2 and 3) seem to interact with the other proteins in the signalling complex(cite). We chose to use a truncated version of the sIL-6R were we only use domain 2 and 3 since we thought this would be easier to express in our chassis.
+        This interaction causes the intracellular parts of the two gp130 proteins
+        to transphosphorylate each other and start a signaling cascade <a href="#gp130" aria-describedby="footnote-label" id="gp130-ref"> </a>.
+        <br>
+        We chose to try out <b>two different designs</b> of our extracellular IL-6 sensing
+        modules. In one design, we link either halves of the intracellular split protein
+        to the extracellular domains of the soluble isoform of gp130 (sgp130) via a
+        transmembrane domain, while secreting a soluble isoform of IL-6R. In this way,
+        the split protein will be reconstituted upon the dimerization of two heterotrimers.<br>
+        <img style="margin-left: 20%;" src="https://static.igem.org/mediawiki/2020/5/5a/T--UCopenhagen--IL6_soluble.png">
+        <figcaption><b>Figure 3: Soluble IL-6 receptor heterotrimerization with sgp130 and IL-6 upon IL-6 binding.</b><br> Binding of IL-6 to sIL-6R leads to heterotrimerization with sgp130, with two trimers forming a dimer complex, thereby initiating the signaling cascade.</figcaption>
+        <br>
+        In the second design we use the association of IL-6 with one gp130 and one sIL-6R in
+        a heterotrimer instead. Here we coupled the N-terminal half of the split protein
+        to sIL-6R and the C-terminal half of the split protein to sgp130.<br>
+        <img style="margin-left: 20%;" src="https://static.igem.org/mediawiki/2020/d/d3/T--UCopenhagen--IL6_kom_nu.png">
+        <figcaption><b>Figure 4: Alternative IL-6 signaling design from figure 3.</b><br> In this design, the N-terminal of the split protein is coupled to sIL-6R and the C-terminal is coupled to sgp130.
+</figcaption>
+        <br>
+        Glycoprotein 130 has six extracellular domains of which the three domains
+        closest to the membrane mainly seem to function by positioning and bringing
+        together the two gp130 proteins in the heterohexameric signaling complex.
+        Since we do not depend on the association of the two gp130 proteins in the
+        second design, we decided to use a truncated version of gp130 where we only
+        use domain 1 to 3 in that design.
+        <br>
+        The sIL-6R has three domains; one Ig-like domain and two fibronectin-like
+        type III domains. Only the two fibronectin-like domains (domain 2 and 3)
+        seem to interact with the other proteins in the signaling complex <a href="#soluble" aria-describedby="footnote-label" id="soluble-ref"> </a>.
+        We chose to use a truncated version of the sIL-6R where we only use domain
+and 3 since we thought this would be easier to express in our chassis.
        </div>
@@ Line 279: / Line 398: @@
          <h3>IL-10</h3>
          <h2>IL-10</h2>
-         The receptors for IL-10 form heterotrimers like the receptors for IL-1.
+         Finally, we chose to look at IL-10, which is an <b>anti-inflammatory interleukin</b>
-         Here, the IL-10R type 1 associated with IL-10, and subsequently recruits
+        released by macrophages after the offending agent has been removed from
-        IL-10R type II. The reason we decided to look into IL-10 is to have a non
+        the site of inflammation. Another difference between IL-10 and the previous
-         acute-phase cytokine in our system. This receptor is also much smaller than
+        two interleukins is that IL-10 is a dimer <a href="#il10" aria-describedby="footnote-label" id="il10-ref"> </a>.
-        the previous two receptors, increasing the chance of correct folding in
+         <br>
-        <em>S. cerevisiae</em>, and is located closer to the membrane compared
+        In IL-10 signaling, the IL-10R type I receptor associates with one domain
-         to the IL-6 receptor for instance.
+        of IL-10, and subsequently recruits the IL-10R type II. Since IL-10 is a
+         dimer, each domain can recruit its own pair of type I and type II receptors <a href="#il10r" aria-describedby="footnote-label" id="il10r-ref"> </a>.
+        <br>
+        Structurally, the receptor is much smaller than the previous two receptors,
+        possibly increasing the chance of correct folding in <em>S. cerevisiae</em>.<br>
+         <img style="margin-left: 20%;" src="https://static.igem.org/mediawiki/2020/9/9b/T--UCopenhagen--IL10.png">
+        <figcaption><b>Figure 5: IL-10R type I association to IL-10R type II upon IL-10 binding.</b><br> Through IL-10 binding to the IL-10R type I receptor, recruitment of IL-10R type II occurs, leading to activation of the signaling pathway. </figcaption>
+        <br>
        </div>
      </div>
      <div class="txt-btwn">
-       <h3>Binding to the Membrane</h3>
+       <h3>Three Biosensor Designs</h3>
-       The extracellular portions of the human interleukin receptors were then
+       <div style="display: flex; width: 100%; justify-content: center;">
-      fused to a transmembrane domain to secure localization to the membrane, as
+        <div style="font-family: Montserrat; font-size: 20px;">Click to go to the design sections!</div>
-      previously mentioned. For this, we researched a lot of different endogenous
+        <br><br>
-      yeast transmembrane domains (hereafter called TMDs) to find the one with
+       </div>
-      the highest predicted localization to the membrane, based on sequence analyses.
+       </div>
-      We ran a sequence analysis on 13 different endogenous single-pass type I
+       Having selected the cytokines to be measured and the receptors used for their
-      transmembrane proteins as part of this endeavor, and using our knowledge
+       detection, we then embarked on establishing different designs for the implementation
-      of the characteristics of the phospholipid bilayer and transmembrane proteins
+       of the biosensor.
-      in general, we came up with our own 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-between those areas. <br>
-      Our candidate had the following sequence of amino acids: <br>
-      <br>
-      <span style="margin: 0 auto"><b>IAGIVIGVVLGVIFILIAILFAFW</b></span>
-      <br><br>
-       And proved to have a higher predicted localization to the membrane than the
-      TMDs we compared it to, as seen here:<br>
-      INSERT PICTURE
-       <br>
-       As this added a level of unpredictability though, we decided to use the TMD
-      Wsc1 for our project design henceforth, as some of our designs build on
-      papers that use the Wsc1 domain (see later). <br>
-      <br>
-      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. This gave rise to our first design: the split-ubiquitin design.
-      <h3>Three Tiers of Biosensor Designs</h3>
-    </div>
      <!-- big big big biggggg tab stuff here -->
          <div class="bigtab">
-           <a href="#Ubiquitin"><img class="bubble" src="https://i.kym-cdn.com/entries/icons/original/000/016/546/hidethepainharold.jpg"></a>
+           <a style="text-decoration: none; color: black;" href="#Ubiquitin"><div style="display: flex; flex-direction: column; justify-content: space-between;">
-           <a href="#TMD""><img class="bubble" src="https://i.kym-cdn.com/entries/icons/original/000/016/546/hidethepainharold.jpg"></a>
+<img class="bubble" style="align-self: center;" src="https://static.igem.org/mediawiki/2020/a/af/T--UCopenhagen--ubiquitin_click.png">
-           <a href="#Alpha"><img class="bubble" src="https://i.kym-cdn.com/entries/icons/original/000/016/546/hidethepainharold.jpg"></a>
+<figcaption style="font-family: Montserrat; font-size: 18px;">Minimal design</figcaption>
+</div></a>
+<div style="width:7.5%;"></div>
+           <a style="text-decoration: none; color: black;" href="#TMD""><div style="display: flex; flex-direction: column; justify-content: space-between;">
+<img class="bubble" style="align-self: center;" src="https://static.igem.org/mediawiki/2020/f/fb/T--UCopenhagen--tev_click.png">
+<figcaption style="font-family: Montserrat; font-size: 18px;">Intermediate design</figcaption>
+</div></a>
+<div style="width:7.5%;"></div>
+           <a style="text-decoration: none; color: black;" href="#Alpha"><div style="display: flex; flex-direction: column; justify-content: space-between;">
+<img class="bubble" style="align-self: center;" src="https://static.igem.org/mediawiki/2020/1/1a/T--UCopenhagen--alpha_click.png">
+<figcaption style="font-family: Montserrat; font-size: 18px;">Advanced design</figcaption>
+      </div></a>
        </div>
-      <div style="display: flex; width: 100%; justify-content: center;"><div style="font-family: Montserrat; font-size: 24px;">Click to go to the design sections!</div></div>
+      <div class="txt-btwn">
+        <h3>Common Parts of Our Biosensor Designs</h3>
+        As previously mentioned, the three interleukin biosensor designs share
+       <b> a common mechanism  of action</b>; the association of two extracellular receptors,
+        resulting in the intracellular association of two halves of a split protein,
+        bound to the extracellular receptors via the same transmembrane domain.
+        Then, they implement different signal processing strategies to achieve
+        different levels of signal processing.
+        <br>
+        <br>
+        <h6 style="font-family: Montserrat; font-size: 20px;">Transmembrane Domain</h6>
+        To ensure localization of the receptor protein fragments to the membrane,
+        we used the <b>Wsc1 endogenous yeast type 1 transmembrane domain</b>, as this had
+        previously been used for protein-protein interaction assays in yeast (see the ubiquitin design).
+        For more information on how we decided on which transmembrane domain to use,
+        see our modeling page.
+        <br>
+        <br>
+        <h6 style="font-family: Montserrat; font-size: 20px;">Transcription Factor</h6>
+        For two of our designs we used the synthetic transcription factor LexA-VP16
+        variant described by Dossani, Z. Y. et al. 2018<a href="#vp16" aria-describedby="footnote-label" id="vp16-ref"> </a>.
+        <br>
+        <br>This consists of the bacterial
+        LexA DNA binding protein, fused with the viral activator domain VP16. A
+        corresponding hybrid promoter with operator regions replaced with sequences
+        that are recognized by LexA is also used, to avoid transcription of yeast-native
+        genes and <b>ensure orthogonality</b>. The promoter we used consisted of six copies of
+        LexO and the eno1 core promoter, in accordance with Rantasalo et. al 2016 <a href="#lex" aria-describedby="footnote-label" id="lex-ref"> </a>.
+      </div>
+      <img src="https://static.igem.org/mediawiki/2020/8/80/T--UCopenhagen--New_VP16.png">
+      <figcaption><b>Figure 6: Release of the synthetic transcription factor, LexA-VP16, leading to gene expression.</b></br> Through release of the synthetic transcription factor, LexA-VP16, in the minimal and intermediate design by cleavage of a flexible link (see below), gene expression is initiated through LexA-VP16 binding to the 6xLexO promoter.</figcaption>
        <div id="Ubiquitin" class="txt-btwn">
-         <h3>Split Ubiquitin</h3>
+         <h3>Split Ubiquitin-Based Reporting</h3>
-         This was the first design we developed, after being introduced to tools
+         This first biosensor design builds on the <b>split-ubiquitin based Membrane
-        for identifying protein-protein interactions by our supervisor. Here we
+        Yeast Two-Hybrid method (MYTH)</b><a href="#myth" aria-describedby="footnote-label" id="myth-ref"> </a>,
-        found the split-ubiquitin based Membrane Yeast Two-Hybrid method (MYTH)
-        <a href="#myth" aria-describedby="footnote-label" id="myth-ref"> </a>,
          where two different proteins of interest are fused to one half of a
          modified ubiquitin molecule each. The modifications made to the two halves
-         of ubiquitin renders them unable to spontaneously bind to each other and
+         of ubiquitin render them unable to spontaneously bind to each other and
-         reconstitute, without first being brought into close proximity of each other
+         reconstitute a functional protein, without first being brought together
          by another protein. This means that if the proteins of interest, that
          the ubiquitin halves are fused to, have a natural affinity for each other
@@ Line 347: / Line 488: @@
          brought closer together by the association on the extracellular side.
          This suits our purposes perfectly, as we already know that the interleukin
-         receptor parts that we’re using have a natural affinity for each other in
+         receptor domains we use have a natural affinity for each other in the
-         the native setting (and of course in the presence of the fitting interleukin).
+         presence of their corresponding interleukin. Thus, by fusing ubiquitin
-        Thus, by fusing ubiquitin to each of our receptors, we know that an extracellular
+        to each of our receptors, we know that an extracellular association will
-        association will result in an intracellular reconstitution of ubiquitin.
+        result in an intracellular reconstitution of ubiquitin.
          <br>
        </div>
        <img src="https://static.igem.org/mediawiki/2020/1/18/T--UCopenhagen--Ubiquitin_design.png">
        <div class="txt-btwn">
-        <br>
          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.
-         As ubiquitin is recognized by deubiquitinating enzymes upon reconstitution,
+         As ubiquitin is recognized by <b>deubiquitinating enzymes</b> upon reconstitution,
          this means that the deubiquitinating enzymes will cleave 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
          its effect.
-        <br>
-        For our purposes, we’ve using a synthetic transcription factor developed
-        by Dossani, Z. Y. et al. 2018. This consists of the bacterial LexA DNA
-        binding protein, fused with the viral activator domain VP16. A corresponding
-        hybrid promoter with operator regions replaced with sequences that are
-        recognized by LexA is also used, to avoid transcription of yeast-native
-        genes and ensure orthogonality <a href="#vp16" aria-describedby="footnote-label" id="vp16-ref"> </a>.
-        <br>
-        INSERT PICCCC
-        <br>
-        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 real signal amplification step. On the other hand, this tried and
-        tested method is a good, secure base to start working from.
-        <br>
-        Under normal circumstances we would’ve tested our design in the wet lab to
-        gauge the importance of amplification at this point, or waited for the dry
-        lab results to see if they confirmed our suspicions, but due to the
-        restrictions in the lab we instead decided to re-think our design from
-        the get-go. This meant that the next step for us would be to make another
-        design, now with preferably more amplification.
-        <br>
-        INSERT ENGNEERING CYCLE FIGURE?
        </div>
+        <img src="https://static.igem.org/mediawiki/2020/1/10/T--UCopenhagen--Ubiquitin2.png">
+        <figcaption><b>Figure 7:  Minimal biosensor design.</b><br> Split ubiquitin complementation leading to release of C-ub bound LexA-VP16 to initiate gene expression.</figcaption><br>
+      <div class="txt-btwn">
+        However, as mentioned in the project description page, the <b>concentrations
+        of interleukins found in sweat are rather low</b>, and as of now, this design
+        has only little  amplification. On the other hand, this tried and tested
+        method was a good, secure base to start working from.
+      </div>
        <div id="TMD" class="txt-btwn">
          <h3>Split TEV Protease and Membrane-bound Transcription Factor</h3>
-         Our intermediate design utilizes the same receptor-system as the ubiquitin-based
+         Our intermediate design utilizes a similar receptor-system to the ubiquitin-based
-         design, but with few modifications. Thanks to our supervisor’s guidance,
+         design. Here, our intracellular split-protein is the <b>split TEV-protease.</b>
-        we were introduced to another kind of split-protein: the split TEV-protease.
+         This is another method to monitor protein-protein interactions, described
-         This was another method developed by Wehr, M. C. et al. In 2006 to monitor
+        by Wehr, M. C. et al in 2006. Here, we again have two engineered inactive
-        protein-protein interactions. Here, we again have two engineered inactive
          halves of the TEV-protease, that only regain activity when coexpressed as
-         fusion constructs with interacting proteins <a href="#tev" aria-describedby="footnote-label" id="tev-ref"> </a>.
+         fusion constructs with interacting proteins<a href="#tev" aria-describedby="footnote-label" id="tev-ref"> </a>. Therefore, we again utilize
-        Therefore, we again utilize the receptor/TMD domains from the previous designs,
+        the receptor/TMD domains from the previous designs, but now each of our
-        but now each of our receptors will be fused to one half of the TEV-protease instead with a flexible linker.
+        receptors will be fused to one half of the TEV-protease instead with a
-        <br>
+        flexible linker.
-        <br>
        </div>
        <img src="https://static.igem.org/mediawiki/2020/8/86/T--UCopenhagen--TEV_design.png">
        <div class="txt-btwn">
-        <br>
          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
+         to the membrane. To this TMD, we’ll fuse the same transcription factor from
-         from the previous design (LexA-VP16), and use the recognition sequence
+         the previous design (LexA-VP16), and <b>use the recognition sequence for the TEV-protease as the linker between the two.</b>
-        for the TEV-protease as the linker between the two. This means that the
+        This means that the TEV-protease, upon reconstitution, will be able to
-        TEV-protease, upon reconstitution, will be able to cleave the transcription
+        cleave the transcription factor and free it into the cytosol. In theory,
-        factor and free it into the cytosol. In theory, the TEV-protease will be
+        the TEV-protease will be able to cut many transcription factors loose,
-        able to cut many transcription factors loose, meaning that one interleukin
+        meaning that one interleukin (by extension of the association of our two
-        (by extension of the association of our two receptors) will result in the
+        receptors) will result in the cleavage of multiple transcription factors
-        cleavage of multiple transcription factors and thus an amplification of the signal.
+        and thus <b>an amplification of the signal.</b>
+      </div>
+        <img src="https://static.igem.org/mediawiki/2020/0/04/T--UCopenhagen--TMDesign.png">
+        <figcaption><b>Figure 8: Intermediate biosensor design.</b><br> Split-TEV protease complementation leading to cleavage of a TEV-recognition sequence near LexA-VP16, thereby releasing LexA-VP16 to initiate gene expression</figcaption>
          <br>
-        <br>
-        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. <br>
-        Simultaneously, we started modeling our pathways in the dry lab.
-      </div>
        <div id="Alpha" class="txt-btwn">
          <h3>Hijacking the Pheromone Pathway</h3>
-         Our last and most ambitious design hinges on hijacking the pheromone pathway
+         Our most <b>ambitious</b> design hinges on hijacking the pheromone pathway in
-         in yeast. We had no doubt that this would give us the most amplification,
+         yeast. This design has the potential to provide a high level of amplification,
-         which is especially important in our case given the low concentrations of
+         which is especially important given the low concentrations of interleukins
-         interleukins in sweat (SOURCE????). The pheromone pathway is XXXXX EXPLAIN HERE OR NO?
+         in sweat <a href="sweat" aria-describedby="footnote-label" id="sweat-ref"> </a>.
-        <br>
+      </div>
-        <br>
+      <img src="https://static.igem.org/mediawiki/2020/8/84/T--UCopenhagen--pheromone_cascade.png">
-         In order to hijack the pheromone pathway, we initially set out to understand
+      <figcaption><b>Figure 9: The endogenous yeast pheromone mating pathway.</b></br> Upon alpha factor (pheromone/ligand) binding to the GPCR, intracellular signaling is initiated, and through activation of the Ste12 transcription factor, expression of genes related to the pheromone pathway is initiated. </figcaption>
-        how the Ste5 scaffold protein was recruited to the membrane by the beta/gamma
+      <br>
-        complex. Soon though, we saw that the interaction between the beta/gamma
+      <div class="txt-btwn">
-        complex and the scaffold was more complicated than previously thought, and
+      <div class="todelt">
-        introducing changes to this step seemed risky (SOURCE about how we don’t
+         <div class="txt-btwn"><div class="format">
-        know what beta/gamma does exactly here). Since the exact mechanism by which
+          Yeast has a <b>G-protein-coupled receptor (GPCR)</b> specific to yeast mating pheromones.
-        the beta/gamma complex recruits Ste5 was unknown to us, we decided to take
+          When a pheromone binds to the receptor, the receptor occupancy stimulates
-        a more conservative approach instead of changing beta/gamma for another
+          the G-alpha subunit of the G-protein to exchange GDP for GTP, and release
-        recruiting maneuvre. Here, we finally thought to introduce an inhibitory
+          the beta and gamma subunits. The released beta and gamma subunits can
-        sequence on beta, that could be removed at will, so as to keep the beta/gamma
+          then recruit the Ste5 scaffold protein to the membrane, starting a
-        complex as the recruiting element. This inhibitory sequence would then,
+          phosphorylation cascade eventually leading to the phosphorylation and
-        building onto the previous design, contain the TEV protease cleavage site,
+          activation of the transcription factor Ste12 and expression of pheromone
-        so an extracellular signal could trigger the TEV protease to cut off the
+          response genes <a href="#phero" aria-describedby="footnote-label" id="phero-ref"> </a>.
-        inhibitory sequence and start the signaling. The next step from here would
+          <br>
-        be to design such an inhibitory sequence, and through talking to our
+          <br>
-        supervisor, we agreed that we should stay in the same conservative vein
+          Since the dissociation of G-alpha from the beta and gamma subunits
-        and look at natural inhibitors of the signaling. Here, the alpha subunit
+          is what drives the pheromone cascade, we decided to design our own
-        of the G protein was an obvious choice, as the alpha subunit binds to
+          switch for this step in the pathway. Building onto the previous TEV
-        beta/gamma in the absence of an extracellular signal, and hinders them
+          protease design, we designed <b>a mutant G-alpha with cleavage sites
-        from recruiting Ste5 and completing the pheromone pathway signaling.
+          from the TEV protease</b> inserted at multiple points, as described on
-        <br>
+          our engineering success page. This meant that the presence of an
-        As a natural inhibitor, G alpha was a great choice for us. <br>
+          interleukin, and ultimately the reconstitution of the TEV protease,
-        <br>
+          would result in the cleavage of G-alpha into multiple fragments. These
-        The next step from here was to find places to insert the TEV protease
+          fragments would then, in theory, dissociate from the beta/gamma subunit,
-        cleavage sites in G alpha. Our goals when doing this was to have a G alpha that
+          freeing it in a similar fashion to the normal signal transduction pathway.
-        <br>
+          The free beta/gamma subunit would then be able to trigger the pheromone
-        <li>
+          pathway and activate a downstream reporter gene. In order to maintain
-           <ol>1. could keep its GTPase activity, so it could be used in other contexts
+          orthogonality, we used the same promoter as previously, and the
-        in the future.</ol>
+          transcription factor <b>LexA-Ste12</b> - a synthetic transcription factor
-          <ol>2. could bind to and exert an inhibitory function on the beta/gamma
+          that can be <b>activated by phosphorylation</b> in the last step of the
-        complex’s ability to recruit Ste5.</ol>
+          phosphorylation cascade in the pheromone pathway.
-          <ol>3. could, once cut by the TEV protease in the presence of a signal,
+          <br>
-        dissociate from beta/gamma again, enabling regular signaling and recruitment of Ste5.</ol>
+          <br>
-        </li>
+          First, we set out to find places to insert the TEV protease cleavage
-         <br>
+          sites in yeast G alpha (GPA1p). Our goals when doing this was to
-        <br>
+          engineer a G alpha that
-        For this, we had many approaches. Our first thought was to look at the yeast
+          <br>
-        G alpha’s sequence, and change very few amino acids in places that already
+           <ol>
-        resembled the TEV recognition site. During this first iteration, we used
+            <li><b>1.</b> could bind to and exert an <b>inhibitory function on the beta/gamma</b> complex’s ability to trigger the signalling pathway cascade.</li>
-        the following recognition site:
+            <li><b>2.</b> could, once cut by the TEV protease in the presence of a signal, <b>dissociate from beta/gamma</b> again, triggering the pathway. </li>
-         <br>
+          </ol>
-        <br>
+         </div></div>
-        EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic and
+         <div class="image">
-        φ is any small hydrophobic or polar residue <a href="#site" aria-describedby="footnote-label" id="site-ref"> </a>.
+          <div style="display: flex; flex-direction: column; justify-content: space-between;">
-        <br>
+            <img src="https://static.igem.org/mediawiki/2020/2/24/T--UCopenhagen--G_alpha_samlet.png">
-        (INSERT PRELIMINARY RESULTS?)
+            <figcaption><b>Figure 10: Advanced biosensor design.</b><br> Split-TEV protease complementation leading to cleavage of a TEV protease cut site in GPA1, thereby initiating gene expression through the yeast pheromone pathway.</b></figcaption>
-        <br>
+          </div>
-        <br>
+         </div>
-        However, we found that introducing different amino acids (albeit with
+         </div>
-        the same properties) in the different cleavage sites would prove to make
+         <div class="txt-btwn">
-        subsequent wet lab troubleshooting harder, as well as change the dry lab
+          Our approach can be seen in more detail under the modeling and engineering success pages.
-        output in unreasonable ways, as the TEV protease would then have different
+          <br><br>
-        efficiencies depending on the used recognition site (INSERT SOURCE DAVID’S
+          To summarize, our last and final design entails:
-        ARTICLE FROM BENCHLING). In order to limit the amount of unknowns, and
+      <ol>
-        to have the same affinity of our TEV protease to our recognition site,
+         <li><b>1.</b> A signaling <b>interleukin</b> reaches our two receptors.</li>
-        we opted to use the same recognition site everywhere; <b>ENLYFQG.</b>
+        <li><b>2.</b> The receptors associate extracellularly, giving rise to the intracellular
-        <br>
+         <b>complementation of a split TEV protease.</b></li>
-         <br>
+        <li><b>3.</b> The now active TEV protease can <b>cut a mutant G alpha</b> protein into smaller
-         Using mutagenesis results from uniprot and other literature (SOURCE????
+         peptide fragments, that’ll dissociate from the beta/gamma complex.</li>
-        May be weird, I’m sleepy) we found the residues that have been found to
+        <li><b>4.</b> The beta/gamma complex can exert its recruiting function on the Ste5
-        give constitutive activity in the pheromone pathway as a result of alpha
+         scaffold protein and recruit it to the membrane, thus <b>triggering the
-        not being able to bind to beta/gamma (SOURCE insert Gladue, D. P. 2008)
+         pheromone cascade</b>, eventually resulting in the phosphorylation of our modified LexA-Ste12 transcription factor.</li>
-        etcetera, and through extensive computer modeling described under our
+        <li><b>5.</b> Our activated, phosphorylated transcription factor LexA-Ste12 will
-        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. For details, check out our
-         <a href="https://2020.igem.org/Team:UCopenhagen/Engineering_Success">engineering success page!</a>
-        <br>
-        <br>
-        So, to summarize, our last and final design entails:
-         <li>
-          <ol>1. A signaling interleukin reaches our two receptors.</ol>
-          <ol>2. The receptors associate extracellularly, giving rise to the intracellular
-         complementation of a split TEV protease.</ol>
-          <ol>3. The now active TEV protease can cut a mutant G alpha protein into smaller
-         peptide fragments, that’ll dissociate from the beta/gamma complex.</ol>
-          <ol>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, eventually resulting in the phosphorylation of our modified LexA-Ste12 transcription factor.</ol>
-          <ol>5. Our activated, phosphorylated transcription factor will
          bind to the synthetic promoter 6xlexo eno1, and result in the transcription and translation of
-         our reporter protein.</ol>
+         our <b>reporter protein.</b></li>
-        </li>
+      </ol>
-        <br>
+      <br>
-        <br>
+    </div>
-        This last and final design has a lot of strengths compared to the two prior
+    <div class="txt-btwn">
-        designs, but also some drawbacks. Of course, changing such a conserved protein
+      <h3>Why Three Designs?</h3>
-        is a great challenge, as any small change could result in great consequences for its
+      This last and final design has a lot of <b>strengths</b> compared to the two prior
-        conformation and normal functions. At the same time, however, this design proved
+      designs, but also some drawbacks. Of course, changing such a conserved
-        to be the best in terms of amplification and sensitivity, as shown through
+      protein is a great challenge, as any small change could result in great
-        our <a href="https://2020.igem.org/Team:UCopenhagen/Model">modeling</a> of
+      consequences for its conformation and normal functions. At the same time,
-        the system compared to the two others.
+      however, this design proved to be <b>the best in terms of amplification</b> and
-        <br>
+      <s>sensitivity</s>, as shown through our modeling of the system compared to the
-        <br>
+      two others.
-        For this reason, and to check whether our dry lab results fit with reality,
+      <br>
-        all three of the abovementioned designs were tested in the wet lab, the results
+      <br>
-        of which can be found <a href="https://2020.igem.org/Team:UCopenhagen/Results">here!</a>
+      For this reason, and to check whether our dry lab results fit with reality,
-        <br>
+      all three of the abovementioned designs were tested in the wet lab, the
-        HOW DO WE SAY THAT WE USED THE ENGINEERING CYCLE PERFECTLY, AND WHERE DO I PUT MY FIGURE???????
+      results of which can be found on the results page.<br><br>
-        <br><br><br><br><br><br><br><br><br><br>
+<div style="margin-left:10%; text-align: center;">
+      <a href="https://2020.igem.org/Team:UCopenhagen/Results"><img style="width: 75%;" src="https://static.igem.org/mediawiki/2020/7/72/T--UCopenhagen--results_link.png"></a></div>
+    </div>
        </div>
        </div>
        </div>
 <!-- inserting our footnotes -->
 <div class="ref_cont">
@@ Line 540: / Line 642: @@
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 <a href="#receptor-ref" aria-label="Back to content">↩</a></li>
-         <li id="organism">Gunde T, Barberis A. Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase. Biotechniques. 2005;39(4):541-549. doi:10.2144/000112011<a href="#organism-ref" aria-label="Back to content">↩</a></li>
+         <li id="rtk">Wintheiser GA, Silberstein P. Physiology, Tyrosine Kinase Receptors. [Updated 2020 Oct 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK538532/
-         <li id="organism">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818708/<a href="#organism-ref" aria-label="Back to content">↩</a></li>
+<a href="#rtk-ref" aria-label="Back to content">↩</a></li>
-         <li id="vp16">https://pubmed.ncbi.nlm.nih.gov/29084380/<a href="#vp16-ref" aria-label="Back to content">↩</a></li>
+        <li id="no_rtk">Gunde T, Barberis A. Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase. Biotechniques. 2005;39(4):541-549. doi:10.2144/000112011 <a href="#no_rtk-ref" aria-label="Back to content">↩</a></li>
-         <li id="tev">https://www.nature.com/articles/nmeth967<a href="#tev-ref" aria-label="Back to content">↩</a></li>
+         <li id="il1">Kaneko N, Kurata M, Yamamoto T, Morikawa S, Masumoto J. The role of interleukin-1 in general pathology. Inflamm Regen. 2019;39:12. Published 2019 Jun 6. doi:10.1186/s41232-019-0101-5 <a href="#il1-ref" aria-label="Back to content">↩</a></li>
-         <li id="site">https://pubmed.ncbi.nlm.nih.gov/15477088/<a href="#site-ref" aria-label="Back to content">↩</a></li>
+        <li id="il1native">Fields James K., Günther Sebastian, Sundberg Eric J. Structural Basis of IL-1 Family Cytokine Signaling. Frontiers in Immunology. 2019;10:1412. doi:10.3389/fimmu.2019.01412<a href="#il1native-ref" aria-label="Back to content">↩</a></li>
+        <li id="antagonists">Garlanda, C., Riva, F., Bonavita, E., Gentile, S., & Mantovani, A. (2013). Decoys and regulatory "receptors" of the il-1/toll-like receptor superfamily. Frontiers in Immunology, 4(JUL), [Article 180]. https://doi.org/10.3389/fimmu.2013.00180
+<a href="#organism-ref" aria-label="Back to content">↩</a></li>
+        <li id="CRP">Del Giudice, Marco & Gangestad, Steven. (2018). Rethinking IL-6 and CRP: Why They Are More Than Inflammatory Biomarkers, and Why It Matters. Brain Behavior and Immunity. 70. 61-75. 10.1016/j.bbi.2018.02.013.<a href="#CRP-ref" aria-label="Back to content">↩</a></li>
+        <li id="gp130">Rose-John S, Scheller J, Elson G, Jones SA. Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer. J Leukoc Biol. 2006 Aug;80(2):227-36. doi: 10.1189/jlb.1105674. Epub 2006 May 17. PMID: 16707558.<a href="#organism-ref" aria-label="Back to content">↩</a></li>
+         <li id="soluble">Kurth I, Horsten U, Pflanz S, Timmermann A, Küster A, Dahmen H, Tacken I, Heinrich PC, Müller-Newen G. Importance of the membrane-proximal extracellular domains for activation of the signal transducer glycoprotein 130. J Immunol. 2000 Jan 1;164(1):273-82. doi: 10.4049/jimmunol.164.1.273. PMID: 10605021.
+<a href="#soluble-ref" aria-label="Back to content">↩</a></li>
+        <li id="il10">Zdanov A, Schalk-Hihi C, Gustchina A, Tsang M, Weatherbee J, Wlodawer A. Crystal structure of interleukin-10 reveals the functional dimer with an unexpected topological similarity to interferon gamma. Structure. 1995 Jun 15;3(6):591-601. doi: 10.1016/s0969-2126(01)00193-9. PMID: 8590020.<a href="#il10-ref" aria-label="Back to content">↩</a></li>
+        <li id="il10r">Mosser DM, Zhang X. Interleukin-10: new perspectives on an old cytokine. Immunol Rev. 2008;226:205-218. doi:10.1111/j.1600-065X.2008.00706.x<a href="#il10r-ref" aria-label="Back to content">↩</a></li>
+        <li id="lex">Rantasalo A, Czeizler E, Virtanen R, Rousu J, Lähdesmäki H, Penttilä M, et al. (2016) Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS ONE 11(2): e0148320. https://doi.org/10.1371/journal.pone.0148320<a href="#lex-ref" aria-label="Back to content">↩</a></li>
+        <li id="myth">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<a href="#myth-ref" aria-label="Back to content">↩</a></li>
+         <li id="tev">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<a href="#tev-ref" aria-label="Back to content">↩</a></li>
+         <li id="sweat">Hladek, Melissa & Szanton, Sarah & Cho, Young-Eun & Lai, Chen & Sacko, Caroline & Roberts, Laken & Gill, Jessica. (2017). Using sweat to measure cytokines in older adults compared to younger adults: A pilot study. Journal of Immunological Methods. 454. 10.1016/j.jim.2017.11.003. <a href="#sweat-ref" aria-label="Back to content">↩</a></li>
+        <li id="phero">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<a href="#phero-ref" aria-label="Back to content">↩</a></li>
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