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Using recently released proteomics data on B. bigelowii and UCYN-A, along with older genomics and transcriptomics data, we have identified a putative list of proteins that are imported into the organelle. This data provides a solid foundation for further research into which proteins are essential, as we suspect many are redundant. Identifying a list of essential host-encoded proteins is crucial to successfully transplanting UCYN-A into a new host.
We have also created a new transcriptome assembly of B. bigelowii based on raw data from previous studies, using improved algorithms. This allowed us to create a new predicted proteome.
We are making all of our omics data available for future iGEM teams along with documentation. </p>
<p></p>Read more on our <ahref="https://2024.igem.wiki/tu-delft/results">results</a> page
<p>Read more on our <ahref="https://2024.igem.wiki/tu-delft/results">results</a> page
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<divclass="h1">Motivation</div>
<p>Being a team from the Netherlands, we have actively followed the <strong>unfolding of the nitrogen crisis</strong> and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with <strong>agricultural exports</strong> being worth 124 billion euros in 2023 alone <ahref="#cite8"style="color: #185A4F;">[8]</a>.</p>
<p>The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 <ahref="#cite9"style="color: #185A4F;">[9]</a> and Stony-Brook 2023 <ahref="#cite10"style="color: #185A4F;">[10]</a> that have tackled similar challenges, alongside a recent publication in Nature in April 2024 <ahref="#cite11"style="color: #185A4F;">[11]</a>.</p>
<divclass="h1">Characterization of UCYN-A transit peptide</div>
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<divclass="h1">Solution</div>
<p>We are motivated by the vision of making <strong>the first step of what could be one of the biggest contributions to sustainable agriculture in the near future</strong>. We believe that the use of the nitroplast's capabilities could lead to more eco-friendly farming practices and help address some of the pressing challenges associated with current fertilization techniques, both in the Netherlands where there is a major nitrogen crisis, and globally where a growing demand for feed crops clashes with a need to reduce greenhouse emissions. Our project aims to harness the power of this organelle to create a <strong>more sustainable and efficient approach to crop cultivation</strong>, ultimately benefiting both the environment and the agricultural industry.</p>
<p>The Nature publication by Coale <em>et al.</em> examines UCYN-A, which evolved from a cyanobacterial species capable of converting N<sub>2</sub> into organic nitrogenous compounds, and its relationship with the marine alga <em>Braadurosphaera bigelowii</em>. It has already been established that UCYN-A and <em>B. bigelowii</em> have a symbiotic relationship, where <em>B. bigelowii</em> functions as a so-called host, and has taken up the UCYN-A bacteria into its cell in a process known as endosymbiosis. <strong>The symbiont, UCYN-A, fixes nitrogen for the host</strong> whereas <em>B. bigelowii</em> supplies organic carbon and a conducive living environment. This paper proved that UCYN-A is not simply a symbiont, but has instead evolved into an organelle for the eukaryotic alga for nitrogen fixation, and is now called the <strong>"nitroplast"</strong><ahref="#cite11"style="color: #185A4F;">[11]</a>.</p>
<p>The discovery of the nitroplast captured our interest - we had considered a project on nitrogen fixation before but failed to see a way in which we could innovate or propose new solutions to the problems previous teams faced. All diazotrophs (bacteria and archaea that fix atmospheric N<sub>2</sub>) use the <strong>enzyme nitrogenase</strong> to fix nitrogen, but the expression of this enzyme presents great difficulties: it is <strong>irreversibly damaged by reacting with oxygen</strong>, while at the same time catalyzing an energetically demanding reaction. Due to this, diazotrophs have evolved very complex mechanisms to couple nitrogen fixation with respiration and/or photosynthesis, which so far has been beyond reach in terms of reproduction by synthetic biologists. The <strong>nitroplast solves this problem, acting as a fully contained compartment within a eukaryote where nitrogen fixation takes place</strong>, utilizing several years of evolutionary optimization.</p>
<p><strong>Symbiotic relationships</strong> between diazotrophs and plants already exist in nature, specifically in crops - legumes have a relationship with rhizobia (bacteria living around the plant root), as do some grass species with other nitrogen-fixers. However, most <strong>other crops do not have anything of the sort</strong>. Besides transgenic nitrogenase expression, the other main avenue currently being explored in nitrogen fixation is the <strong>engineering of external symbiosis</strong> between diazotrophs and other plants. However, this poses a challenge in replicating fragile extracellular signaling pathways and physical conditions that are dependent on the plant species' roots, as well as potential containment issues.</p>
<p>Replicating endosymbiosis, while more ambitious than root-bacteria symbiosis, <strong>ensures by design that cell and organelle will work tightly together</strong>, preventing the difficulties associated with either root-dependence or nitrogenase expression. Our ideal <strong>long-term goal would be to introduce this organelle into crops</strong>. By doing this, it may be possible to <strong>reduce the reliance on synthetic fertilizers</strong>, thereby lowering environmental impact of their production and use, and enhancing sustainability in agriculture. This potential for positive change inspired our group to explore this innovative solution further.</p>
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<divclass="h1">Our Approach</div>
<p>One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. <strong>To this end, we first need to better study the nitroplast, how it interacts with the host organism and how it could be potentially introduced into other cells</strong>.</p>
<p>It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, <em>B. bigelowii</em>, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent <ahref="#cite11"style="color: #185A4F;">[11]</a>. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified <ahref="#cite11"style="color: #185A4F;">[11]</a>. Our first aim was to employ bioinformatics analyses to identify the characteristic <strong>motifs required for a protein to be imported by UCYN-A</strong>. For this, we made use of host (<em>B. bigelowii</em>) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale <em>et al.</em>. <strong>We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</strong>.</p>
<p>To understand the functioning of the UCYN-A import mechanism, we attempted to <strong>identify the proteins involved in translocating</strong> host-encoded proteins into UCYN-A. First, we located genes in the host genome that are potentially involved in the translocation, based on their similarity to proteins in other import mechanisms such as from <em>Paulinella chromatophora</em> (UCYN-A analogue for photosynthesis). Potential chaperones analogous to heat-shock proteins were also included in the search. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. We then followed this by <strong>obtaining the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and used <strong>docking</strong> tools to select candidate proteins likely to bind the previously identified transit motifs.</p>
<p>In addition to <em>in silico</em> experiments, we also aimed to investigate the transport mechanisms of UCYN-A <em>in vivo</em>. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast <em>S. cerevisiae</em> and the green alga <em>C. reinhardtii</em>. The initial <em>in vivo</em> characterization of the UCYN-A transport system involved <strong>examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms</strong> to test whether uTP would have any unexpected effect on cell viability and would not target any other organelle. To this end, we designed vectors, cloned them using Gibson-assembly, transformed bacteria, purified expression plasmids, and transformed those into <em>S. cerevisiae</em> and <em>C. reinhardtii</em>. We expressed uTP-tagged fluorescent proteins, together with controls targeting other organelles, and localization was assessed using fluorescence microscopy. Our preliminary results indicate that uTP did not target any other organelle and did not lead to alterations in cell morphology.</p>
<p>Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <em>S. cerevisiae</em> using either <em>E. coli</em> or <em>S. elongatus</em><ahref="#cite12"style="color: #185A4F;">[12]</a>. Another study successfully inserted <em>Azotobacter</em> strains into <em>C. reinhardtii</em><ahref="#cite13"style="color: #185A4F;">[13]</a>. Building on this research, we initially aimed to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <em>C. reinhardtii</em> and <em>S. cerevisiae</em> as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, <em>E. coli</em>, and refined a protocol for its fusion with <em>S. cerevisiae</em>.</p>
<p>We obtained <em>B. bigelowii</em> as a gift from Dr. Kyoko Hagino (Kochi University, Japan) and cultured it according to the protocol. For the transplantation of the nitroplast into a different model organism, we were also required to isolate this organelle from its host. Our project also implemented the protocol for nitroplast isolation from its host, according to the protocol and suggestions provided by Dr. Tyler Coale (University of California, San Diego, USA). Isolated UCYN-A could potentially be used for PEG fusion with other eukaryotes.</p>
<p>Finally, we have also wondered how our project would affect society. For this, our human practices team has actively worked to understand the possible consequences of our project. Also, wider acceptance of our idea goes hand in hand with educating the general population. Last but not least, we have also assessed the potential economic benefits of our idea and the omission of fertilizer use in agriculture. For this, we made an <strong>economic analysis</strong> and business plan.</p>
<p>Our project <strong>lays the foundation for the transplantation of nitroplast</strong> into eukaryotic hosts. The emergence of nitrogen-fixing plants could lead to a significant drop in the demand for fertilizers, and consequently in both carbon emissions and nitrogen pollution.</p>
<divclass="h1">Peptide binding model</div>
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<divclass="h1"><em>B. bigelowii</em> on list A1</div></div>
