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-      <p>To lay the foundation for a new avenue of research into engineering nitrogen-fixing endosymbionts, we investigated protein transport to B. bigelowii’s symbiotic partner UCYN-A (nitroplast) and implemented proof-of-principle endosymbiosis experiments. 
+      <p>To lay the foundation for a new avenue of research into engineering nitrogen-fixing endosymbionts, we investigated protein transport to <em>B. bigelowii</em> ’s symbiotic partner UCYN-A (nitroplast) and implemented proof-of-principle endosymbiosis experiments. 
       </p>
 
 
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             <p>The majority of proteins within mitochondria and chloroplasts are nuclear-encoded – they are expressed by the host and are imported into the organelle. Proteins meant for the organelle are usually marked by a targeting sequence at one end, also known as a transit peptide, which directs the protein to its destination after which it is cleaved. </p>
             <p>This is no different with UCYN-A: Coele et al <a href="#cite1" style="color: #185A4F;">[1]</a> in their 2024 study used proteomics to find proteins encoded by the host and imported into the nitroplast. Upon examining these protein sequences, they noticed that many of them possess characteristics of organellar import – most of them possess a C-terminal 120 amino acid extension compared to their orthologues. This extension is reminiscent of targeting sequences known to exist in mitochondrial <a href="#cite2" style="color: #185A4F;">[2]</a> and chloroplastic <a href="#cite3" style="color: #185A4F;">[3]</a> imported proteins. They termed the putative targeting sequence uTP (UCYN-A Transit Peptide, with lowercase “u” to differentiate it from uridine triphosphate).
             </p>
-            <p>Our investigations began with an in-depth computational analysis of B. bigelowii’s proteome <a href="#cite1" style="color: #185A4F;">[1]</a>, <a href="#cite7" style="color: #185A4F;">[7]</a> to identify potential signals marking proteins for import into UCYN-A. Based on these results, we designed fluorescent protein-transit peptide constructs for expression in model organisms to show that the identified signals indeed localize to UCYN-A. To pave the way for transplanting the nitroplast into new organisms, we also explored the feasibility of physically inserting UCYN-A into a new host by attempting cell fusion experiments. Furthermore, we successfully established a culture of B. bigelowii and tested a new protocol for isolating UCYN-A. These experiments collectively aim to elucidate the mechanisms of UCYN-A's endosymbiotic relationship and lay the groundwork for future engineering of nitrogen-fixing symbionts into new host organisms.</p>
+            <p>Our investigations began with an in-depth computational analysis of <em>B. bigelowii</em> ’s proteome <a href="#cite1" style="color: #185A4F;">[1]</a>, <a href="#cite7" style="color: #185A4F;">[7]</a> to identify potential signals marking proteins for import into UCYN-A. Based on these results, we designed fluorescent protein-transit peptide constructs for expression in model organisms to show that the identified signals indeed localize to UCYN-A. To pave the way for transplanting the nitroplast into new organisms, we also explored the feasibility of physically inserting UCYN-A into a new host by attempting cell fusion experiments. Furthermore, we successfully established a culture of <em>B. bigelowii</em> and tested a new protocol for isolating UCYN-A. These experiments collectively aim to elucidate the mechanisms of UCYN-A's endosymbiotic relationship and lay the groundwork for future engineering of nitrogen-fixing symbionts into new host organisms.</p>
 
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               <img src="https://static.igem.wiki/teams/5054/graphical-abstract.png" alt="Fig 1: Graphical overview of the experiment plan.">
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             <p>For in vivo characterization, we constructed candidate uTP sequences by concatenating the consensus sequences of discovered motif patterns. The uTP sequence classifiers were used to select the correct motifs for the fluorescent proteins we planned to use, mVenus and mNeonGreen. The two sequences with the highest confidence values (uTP1 and uTP2) were selected for in vivo experiments. These sequences were also submitted to the Parts Registry.</p>
-            <p>To further validate the constructed sequences, their predicted structure was examined. Structural prediction was performed on all 206 selected uTP-containing B. bigelowii proteins, to uncover the 3D conformation of uTP. The predicted structures were aligned and a consensus structure was created by averaging the aligned regions. This revealed a highly-conserved (stdev per residue position < 1.8Å) structural region with 2 alpha-helices arranged into a U-bend (Fig 5). The structure of constructed mNeonGreen and mVenus + uTP1, uTP2 sequences was predicted and the consensus structure aligned onto them, yielding good alignment (RMSD<=4.0Å), confirming that our constructs will likely behave similar to native uTP-containing proteins.</p>
+            <p>To further validate the constructed sequences, their predicted structure was examined. Structural prediction was performed on all 206 selected uTP-containing <em>B. bigelowii</em> proteins, to uncover the 3D conformation of uTP. The predicted structures were aligned and a consensus structure was created by averaging the aligned regions. This revealed a highly-conserved (stdev per residue position < 1.8Å) structural region with 2 alpha-helices arranged into a U-bend (Fig 5). The structure of constructed mNeonGreen and mVenus + uTP1, uTP2 sequences was predicted and the consensus structure aligned onto them, yielding good alignment (RMSD<=4.0Å), confirming that our constructs will likely behave similar to native uTP-containing proteins.</p>
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               <img src="https://static.igem.wiki/teams/5054/structures.svg" alt="Fig 1: Graphical overview of the experiment plan." style="background: white;"> 
             <figcaption>Figure 5: Structural predictions. (a) Consensus structure of all uTP sequences extracted from UCYN-A imported proteins. (b) Consensus structure of all uTP sequences with charged residues shown (red=negative, blue=positive). (c) Consensus structure aligned onto uTP1 + mNeonGreen construct (RMSD=4.00Å). (d) Consensus structure aligned onto uTP2 + mNeonGreen construct (RMSD=3.77Å).            </figcaption>
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             <div class="h" id="four">
               <div class="h2"><em>in vivo</em> uTP characterization</div>
               <div class="h3"><strong>Expression of uTP tagged by Fluorescent proteins</strong></div>
-              <p>The ultimate goal with the uTP sequences we identified is to understand and confirm whether they are indeed responsible for protein import into UCYN-A. Conventional methods to check this would require a toolbox for genetic manipulation of B. bigelowii, not yet available and beyond the scope of this project. We therefore opted for using 2 model eukaryotes for further research on uTP’s behavior, namely C. reinhardtii and S. cerevisiae, and designed an experiment to confirm uTP’s function without modifying B. bigelowii.</p>
+              <p>The ultimate goal with the uTP sequences we identified is to understand and confirm whether they are indeed responsible for protein import into UCYN-A. Conventional methods to check this would require a toolbox for genetic manipulation of <em>B. bigelowii</em>, not yet available and beyond the scope of this project. We therefore opted for using 2 model eukaryotes for further research on uTP’s behavior, namely C. reinhardtii and S. cerevisiae, and designed an experiment to confirm uTP’s function without modifying <em>B. bigelowii</em>.</p>
               <p>We worked off of a <em>Saccharomyces</em> and a <em>Chlamydomonas</em> backbone, pUDE1311 and pOpt2-mVenusBle respectively, in order to design constructs expressing 
               fluorescent proteins (FP) tagged by known transit peptides as well as uTP. Unmodified, pUDE1311 expresses ymNeongreen and pOpt2-mVenusBle expresses mVenus, a YFP analogue; Both express AmpR for selection on <em>E.coli</em>, and while pOpt expresses a Zeocin resistance gene for selection on <em>C. reinhardtii</em> pUDE expresses URA3 for auxotrophic selection on <em>S. cerevisiae</em> CEN.PK 113-5D, an strain with uracil knockout. We designed 2 constructs for expression in our yeast and 3 in our algae. For our yeast, one construct had uTP inserted in the C-terminus of ymNeongreen and the other had MTS1, a mitochondrial transit peptide <a href="#cite2" style="color: #185A4F;">[2]</a>, inserted in the N-terminus of the fluorescent protein.
               For our algae, one construct had uTP inserted in the C-terminus of mVenus, while the two others had a chloroplastic (cTP, <a href="#cite3" style="color: #185A4F;">[3]</a>) and a mitochondrial transit peptide (mTP, <a href="#cite11" style="color: #185A4F;">[11]</a>) respectively, both inserted in the N-terminus of mVenus. Plasmid maps for our constructs and vectors can be found in our Materials and Methods page.
@@ -183,7 +183,7 @@
               <div class="h2">UCYN-A isolation and fusion</div>
               <div class="h3"><strong><em>B. bigelowii</em> culture and UCYN-A isolation</strong></div>
 
-              <p>Thanks to the generous help of Dr. Kyoko Hagino, a pioneer in research into B. bigelowii, we obtained a culture of B. bigelowii FR-21 <a href="#cite1" style="color: #185A4F;">[1]</a>. This species is known to be difficult to work with, however, we were able to find the optimal conditions and grow it in our lab in Delft, establishing, to our knowledge, the first B. bigelowii culture in Europe. We followed Kyoko’s advice when deciding on our culture conditions, which can be found in our Materials and Methods section.</p>  
+              <p>Thanks to the generous help of Dr. Kyoko Hagino, a pioneer in research into <em>B. bigelowii</em>, we obtained a culture of <em>B. bigelowii</em> FR-21 <a href="#cite1" style="color: #185A4F;">[1]</a>. This species is known to be difficult to work with, however, we were able to find the optimal conditions and grow it in our lab in Delft, establishing, to our knowledge, the first <em>B. bigelowii</em> culture in Europe. We followed Kyoko’s advice when deciding on our culture conditions, which can be found in our Materials and Methods section.</p>  
               <div class="img-pagestyle">
                 <img src="https://static.igem.wiki/teams/5054/bbigelowii.png" alt="Fig 1: Graphical overview of the experiment plan.">
               <figcaption>Figure 11: <em>Braarudosphaera bigelowii</em>, imaged at 1000X magnification on xenic culture medium.
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               </p>
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                 <img src="https://static.igem.wiki/teams/5054/bbigelowii.png" alt="Fig 1: Graphical overview of the experiment plan.">
-              <figcaption>Figure 12: Flow cytometry plot and PCR result for the three populations we identified on B.bigelowii lysate.
+              <figcaption>Figure 12: Flow cytometry plot and PCR result for the three populations we identified on <em>B. bigelowii</em> lysate.
               </figcaption>
               </div>
 
@@ -234,7 +234,7 @@
                 <li id="cite4"> Chatzi, K. E., Sardis, M. F., Tsirigotaki, A., Koukaki, M., Šoštarić, N., Konijnenberg, A., Sobott, F., Kalodimos, C. G., Karamanou, S., & Economou, A. (2017). Preprotein mature domains contain translocase targeting signals that are essential for secretion. Journal of Cell Biology, 216(5), 1357–1369. <a href="https://doi.org/10.1083/JCB.201609022" style="color:#185A4F;">https://doi.org/10.1083/JCB.201609022</a></li>
                 <li id="cite5"> Mehta, A. P., Supekova, L., Chen, J., Pestonjamasp, K., Webster, P., Ko, Y., Henderson, S. C., McDermott, G., Supek, F., & Schultz, P. G. (2018). Engineering yeast endosymbionts as a step toward the evolution of mitochondria. Proceedings of the National Academy of Sciences, 115(46), 11796–11801. <a href="https://doi.org/10.1073/pnas.1813143115" style="color:#185A4F;">https://doi.org/10.1073/pnas.1813143115</a></li>
                 <li id="cite6"> Kunová, N., Ondrovičová, G., Bauer, J. A., Bellová, J., Ambro, Ľ., Martináková, L., Kotrasová, V., Kutejová, E., & Pevala, V. (2017). The role of Lon-mediated proteolysis in the dynamics of mitochondrial nucleic acid-protein complexes. Scientific Reports, 7(1). <a href="https://doi.org/10.1038/s41598-017-00632-8" style="color:#185A4F;">https://doi.org/10.1038/s41598-017-00632-8</a></li>
-                <li id="cite7"> Suzuki, S., Kawachi, M., Tsukakoshi, C., Nakamura, A., Hagino, K., Inouye, I., & Ishida, K. (2021b). Unstable Relationship Between Braarudosphaera bigelowii (= Chrysochromulina parkeae) and Its Nitrogen-Fixing Endosymbiont. Frontiers in Plant Science, 12. <a href="https://doi.org/10.3389/fpls.2021.749895" style="color:#185A4F;">https://doi.org/10.3389/fpls.2021.749895</a></li>
+                <li id="cite7"> Suzuki, S., Kawachi, M., Tsukakoshi, C., Nakamura, A., Hagino, K., Inouye, I., & Ishida, K. (2021b). Unstable Relationship Between <em>Braarudosphaera bigelowii</em> (= Chrysochromulina parkeae) and Its Nitrogen-Fixing Endosymbiont. Frontiers in Plant Science, 12. <a href="https://doi.org/10.3389/fpls.2021.749895" style="color:#185A4F;">https://doi.org/10.3389/fpls.2021.749895</a></li>
                 <li id="cite8"> Albiniak, A. M., Baglieri, J., & Robinson, C. (2012). Targeting of lumenal proteins across the thylakoid membrane. Journal of Experimental Botany, 63(4), 1689–1698. <a href="https://doi.org/10.1093/JXB/ERR444" style="color:#185A4F;">https://doi.org/10.1093/JXB/ERR444</a></li>
                 <li id="cite9"> Peeters, N., & Small, I. (2001). Dual targeting to mitochondria and chloroplasts. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1541(1–2), 54–63. <a href="https://doi.org/10.1016/S0167-4889(01)00146-X" style="color:#185A4F;">https://doi.org/10.1016/S0167-4889(01)00146-X</a></li>
                 <li id="cite10"> Ojala and Garriga. <a href="https://www.jmlr.org/papers/volume11/ojala10a/ojala10a.pdf" style="color:#185A4F;">Permutation Tests for Studying Classifier Performance.</a> J. Mach. Learn. Res. 2010</li>