results.html 10.04 KiB
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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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<span>Results</span>
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<div onclick="goTo(document.getElementById('one'))"><span id="subtitle1" style="color: #62D881;"><strong>DRY LAB</strong></span></div>
<div onclick="goTo(document.getElementById('two'))"><span id="subtitle2" style="color: #62D881;"><strong>WET LAB</strong></span></div>
<div onclick="goTo(document.getElementById('three'))"><span id="subtitle3" style="color: #62D881;">uTP-FP expression </div>
<div onclick="goTo(document.getElementById('four'))"><span id="subtitle4" style="color: #62D881;">UCYN-A fusion</div>
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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.
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<div class="h1"><strong>DRY LAB</strong></div>
<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 [1] 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 [2] and chloroplastic [3] imported proteins. They termed the putative targeting sequence uTP (UCYN-A Transit Peptide, with lowercase “u” to differentiate it from uridine triphosphate).
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<p>Our investigations began with an in-depth computational analysis of B. bigelowii’s proteome [1, 7] 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>
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<img src="https://static.igem.wiki/teams/5054/graphical-abstract.png" alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Fig 1: Graphical overview of the experiment plan. </figcaption>
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<div class="h2">The UCYN-A transit peptide sequence</div>
<p>If, like other organelles, UCYN-A relies on proteins imported from the host for normal functioning, characterizing the import system and the targeting sequence is essential before transplanting the organelle into a new host organism. Building upon the work of Coale et al. [1], we aimed to advance the understanding of uTP by identifying its precise sequence.</p>
<p>Starting from the raw proteomics data from [1], we selected 368 proteins expressed by the host and significantly enriched in UCYN-A and performed multiple sequence alignment (MSA). Using the alignment we identified a strongly conserved C-terminal region in many of the imported proteins similar to that reported by [1]. We selected a subset of 206 proteins with highly similar (>60% sequence identity) C-terminal alignments, indicating that these are likely to contain uTP, </p>
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<img src="https://static.igem.wiki/teams/5054/msa.png" alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Fig 2: Multiple sequence alignment (MSA) of all UCYN-A enriched sequences. The strongly aligned C-terminal region is highlighted (positions 880-1010 in the alignment)
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<p>Motif analysis confirmed findings similar to [1], revealing 8 conserved motifs in the C-terminal region (Fig 2). Further investigation of motif co-occurrence and relative positioning uncovered common patterns: two motifs consistently appeared near the start of the C-terminal region at fixed positions, followed by various combinations of the remaining motifs. This arrangement is reminiscent of a potential sub-organellar localization mechanism, where the initial two motifs could target UCYN-A, while subsequent motifs may specify localization within the endosymbiont, as is the case with chloroplast targeting, where a bipartite N-terminal targeting sequence specifies stromal and thylakoidal localization. More research is needed however to investigate this hypothesis.</p>
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<img src="https://static.igem.wiki/teams/5054/msa.png" alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 3: 5 uTP variations discovered among UCYN-A imported sequences. The left panel shows the relative position of conserved motifs in the different uTP variations, relative to motif #1, which was present in all examined sequences.
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<p>We investigated the relationship between transit peptide (uTP) sequences and the functional core of proteins, known as the mature domain. The mature domain is the part of a protein that remains after the transit peptide is cleaved off and performs the protein's primary function. Given the observed diversity in uTP sequences, understanding their connection to specific mature domains is crucial for designing effective uTP constructs for future experiments. Certain uTP sequences may only be compatible with specific proteins, so to explore potential correlations between uTP motif patterns and mature domain sequences, we trained classifiers to predict the appropriate uTP sequence (by predicting the correct combination of motifs) based on a given mature domain sequence. The classifiers were evaluated using a permutation test [10], with 3 of them yielding statistically significant results (p < 0.05) (Fig 4).</p>
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<img src="https://static.igem.wiki/teams/5054/msa.png" alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 4: Classification results of 4 different classifiers trained to predict uTP sequences based on mature domains.
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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>
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<div class="h1"><strong>WET LAB</strong></div>
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<div class="h2">Expression of uTP tagged by Fluorescent proteins</div>
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<div class="h2">UCYN-A isolation and fusion</div>
<div class="h3"><em>B. bigelowii</em> culture and UCYN-A isolation</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 [1]. 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>
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<img src="https://static.igem.wiki/teams/5054/msa.png" alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption><em>Braarudosphaera bigelowii, imaged at 1000X magnification on xenic culture medium.</em>
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<div class="h3">PEG fusion</div>
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