<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>
<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>
<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>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 in <ahref="#cite11"style="color: #185A4F;">[11]</a>. <ahref="results#two"style="color: #185A4F;">We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2</a>.</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>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 <ahref="results#four"style="color: #185A4F;">fluorescence microscopy</a>. 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 <ahref="results#five"style="color: #185A4F;">fusion with <em>S. cerevisiae</em></a>.</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>Finally, we have also wondered how our project would affect society. For this, our <ahref="human-practices"style="color: #185A4F;">human practices</a> 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 <ahref="entrepreneurship"style="color: #185A4F;"><strong>economic analysis</strong> and business plan</a>.</p>
<liid="cite11">Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. Coale T.H. and T. Nishimura. “Nitrogen-fixing organelle in a marine alga”. In: Science 384 (2024), pp. 217–222.</li>
<liid="cite11">Coale T.H. Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. and T. Nishimura. “Nitrogen-fixing organelle in a marine alga”. In: Science 384 (2024), pp. 217–222.</li>
<liid="cite12">Angad P. Mehta et al. “Engineering yeast endosymbionts as a step toward the evolution of mitochondria”. In: Proceedings of the National Academy of Sciences of the United States of America 115.46 (Nov. 2018), pp. 11796–11801. issn: 10916490. doi: 10.1073/PNAS.1813143115/SUPPL{\ _ }FILE/PNAS .1813143115 . SM02 . MP4. url: <ahref="https://www.pnas.org/doi/abs/10.1073/pnas.1813143115"style="color:#185A4F;">https://www.pnas.org/doi/abs/10.1073/pnas.1813143115</a></li>
<liid="cite13">N.H. Nghia et al. “Uptake of Azotobacters by Somatic Fusion of Cell-wall Mutants of Chlamydomonas reinhardii”. In: Biochemie und Physiologie der Pflanzen 181.5 (Jan. 1986), pp. 347–357. issn: 0015-3796. doi: 10.1016/S0015-3796(86)80008-7.</li>
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<!-- <h1>NitroBLAST: Laying the foundation for nitrogen fixation.</h1>
<h2>Background</h2>
<p>The Netherlands has been facing a pressing <strong>nitrogen crisis</strong> for several years. This crisis is largely attributed to the <strong>agriculture sector</strong>, with over 80% of ammonia (a nitrogenous compound) emissions coming from manure <a href="#cite1">[1]</a> and chemical fertilizers <a href="#cite2">[2]</a>. The over-use of fertilizers has a detrimental effect on the environment through the deposition of excess nitrogen oxides and ammonia in the ground, excessively enriching the environment with nutrients promoting uncontrolled plant and algal growth, or eutrophication, a form of nutrient imbalance <a href="#cite3">[3]</a> that negatively impacts the local biodiversity. This highlights the need of the hour: a solution for <strong>increasing global food supply while maintaining environmental standards</strong>.</p>
<h2>Nitrogen Crisis in the Netherlands </h2>
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<img src="https://static.igem.wiki/teams/5054/europe-global-fertilizer-and-manure-version-1-nitrogen-in-manure-production-6172666199.jpg" alt='Farmer protests in the Netherlands [9].' width=80% height = auto/>
<figcaption>Figure 1: Nitrogen manure production in kilograms/hectare in the Netherlands in 2010 <a href="#cite8">[8]</a>.</figcaption>
</figure>
<p>
The Nitrogen Action Programme, introduced by the Dutch government in 2015, aimed at reducing nitrogen deposition, was deemed <strong>insufficient</strong> in 2019 by the council of state. This declaration restricted the building of new residential areas, until the nitrogen emissions were compensated for, further augmenting the ongoing housing crisis of the Netherlands <a href="#cite1">[1]</a>. This emphasizes the urgency of addressing this crisis. <br> <br>
On the other hand, to combat global hunger, an increase in global food production is of the essence. This is addressed through the increase in crop yield, which is possible due to the Haber-Bosch process of fertilizer production, where elemental nitrogen is converted into ammonia. <strong>Over-fertilization</strong> and its direct and indirect impact on the environment make agriculture the second leading contributor to short-term <strong>increases in global surface temperature</strong> <a href="#cite4">[4]</a>.
</p>
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<p>
In 2022, Dutch agriculture lost 74% (312,000 tons) of the nitrogen it spread as manure and synthetic fertilizer to the air and soil. Synthetic fertilizer production alone is also the cause of nearly <strong>2% of global CO<sub>2</sub> emissions</strong> <a href="#cite5">[5]</a>. In addition to <strong>water pollution</strong> by leakage of nitrate, <strong>air pollution</strong> due to the conversion to N<sub>2</sub>O leads to a global greenhouse effect equivalent to 10% of that caused by the increase in atmospheric CO<sub>2</sub> <a href="#cite6">[6]</a>. For staple crops like cereals and maize, <strong>up to 40% of a farm’s operating cost is spent purchasing fertilizer</strong> <a href="#cite4">[4]</a>. Rising prices for fertilizer have been one of the problems leading to farmers' protests in Europe, and efforts to reduce nitrogen emissions in the Netherlands have been met with its own wave of protests <a href="#cite7">[7]</a>. <br> <br>
Altogether, there is a clear and urgent need for an alternative environmental-friendly solution to the nitrogen problem. This can not only make a huge impact on the Netherlands, but also globally, by enabling a sustainable and food secure future.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5054/farmer-protests-in-the-hague-the-netherlands.jpg" alt='Farmer protests in the Netherlands [9].' width="400" height = "300"/>
<figcaption>Figure 2: Farmer protests in the Netherlands <a href="#cite9">[9]</a>.</figcaption>
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<h2>Motivation </h2>
<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 <a href="#cite10">[10]</a>. The Netherlands is also considered one of the <strong>front runners in terms of food and agriculture technology</strong>. The world's first lab-grown meat burger was a Dutch invention, introduced in 2013 <a href="#cite11">[11]</a>. Given our leadership in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 <a href="#cite12">[12]</a> and Stony-Brook 2023 <a href="#cite13">[13]</a> that have tackled similar challenges, alongside a recent publication in Nature in April <a href="#cite14">[14]</a>.</p>
<h2>Solution</h2>
<p>
The Nature publication by Coale <i>et al.</i> examines UCYN-A, a cyanobacterial species capable of converting N<sub>2</sub> into organic nitrogen, and its relationship with the marine algae <i>Bradurosphera bigelowii</i>. It has already been established that UCYN-A and <i>B. bigelowii</i> have a symbiotic relationship, where <i>B. bigelowii</i> 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 <i>B. bigelowii</i> supplies organic carbon and a conducive living environment. This paper proved that UCYN-A is not a common symbiont, but has instead evolved into a eukaryotic organelle for nitrogen fixation, termed the <strong>"nitroplast"</strong> <a href="#cite14">[14]</a>. <br> <br>
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 millions of years of evolutionary optimization. <br> <br>
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 the 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. <br> <br>
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 not-so-distant 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. <br> <br>
</p>
<figure>
<img src="https://static.igem.wiki/teams/5054/igemprojectdescriptionfigure1.png" alt='FEvolution of the nitroplast from the integration of the endosymbiotic UCYN-A bacteria into a eukaryotic.' width=100% height = auto/>
<figcaption>Figure 3: Evolution of the nitroplast from the integration of the endosymbiotic UCYN-A bacteria into a eukaryotic <i>B. bigelowii</i> cell <a href="#cite14">[14]</a>.</figcaption>
</figure>
<h2>Our approach: NitroBLAST</h2>
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<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. To this end, we need to better study the nitroplast and how it could be introduced into other cells. <br> <br>
Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in <i>S. cerevisiae</i> using <i>E. coli</i> and <i>S. elongatus</i> <a href="#cite15">[15]</a>. Another study successfully inserted <i>Azotobacter</i> strains into <i>C. reinhardtii</i> <a href="#cite16">[16]</a>. Building on this research, we aim to <strong>develop a reliable protocol for transplanting a nitroplast</strong> into <i>C. reinhardtii</i> and <i>S. cerevisiae</i>, as a <strong>proof-of-concept</strong> for transplantation into other eukaryotes. We will use polyethylene glycol (PEG) fusion protocols, starting out with analogous bacteria to UCYN-A (<i>Azotobacter</i> genus and <i>Cyanothece</i> ATCC51142, the closest free-living relative of UCYN-A). <br> <br>
It has been discovered that several essential UCYN-A proteins are expressed in the host, <i>B. bigelowii</i>, and imported into the symbiont, not unlike chloroplasts and mitochondria, though to a lesser extent <a href="#cite14">[14]</a>. Many of these proteins possess a C-terminal extension known as the “uTP” (UCYN-A Transit Peptide) <a href="#cite14">[14]</a>. We first aim to use bioinformatic analysis to identify the characteristic <strong>motifs required for a protein to be imported into UCYN-A</strong>. For this, we will make use of host and nitroplast's genome data as well as the proteomics data published by Coale <i>et al.</i>. <br><br>
To understand the functioning of the UCYN-A import mechanism, we will attempt to <strong>identify the proteins involved in translocating</strong> host-encoded proteins into UCYN-A. First, we will locate genes in the host genome which are potentially involved in the translocation based on their similarity to proteins in other import mechanisms such as the <i>Paulinella chromatophora</i> (UCYN-A analogue for photosynthesis) protein import or chaperones that seem analogous to heat-shock proteins. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. Following this, we will <strong>obtain the tertiary structure of all candidate proteins</strong> using a structure prediction tool, and use <strong>docking</strong> tools to select ones that are likely to bind the previously identified transit motifs. <br> <br>
The initial <i>in vivo</i> characterization of the UCYN-A transport system will involve <strong>examining the expression and localization of the UCYN-A transit peptides in the eukaryotic model organisms</strong> <i>S. cerevisiae</i> and <i>C. reinhardtii</i> to test for interference by cellular processes. To this end, uTP-tagged fluorescent proteins will be expressed in <i>S. cerevisiae</i> and <i>C. reinhardtii</i>, and the constructs will be confirmed using fluorescence microscopy. <br><br>
Following up on this, we will <strong>test our candidate chaperone proteins</strong> by expressing them and recreating their interaction with uTP-tagged proteins in our model organisms, in order to demonstrate that the import mechanism can be reproduced outside of <i>B. bigelowii</i>. This will involve constructing plasmids to express fluorescently tagged chaperones and observing colocalization with transit peptide-tagged fluorescent proteins. <br><br>
Finally, while the nitroplast could significantly reduce the need for nitrogen fertilizers, it would also consume energy from its host. Although photosynthesis should supply the plant with sufficient energy for nitrogen fixation, this energy expenditure might impact the growth rate or yield of crops. To assess the potential consequences of nitrogen-fixing staple crops, we will use <strong>metabolic models</strong> to predict effects on growth rate, and <strong>economic models</strong> to link crop yields to farmers’ budgets and profits. <br> <br>
Our project <strong>lays the foundation for the transplantation of nitroplast</strong> into their algal hosts, allowing for the creation of nitrogen-fixing eukaryote strains. The emergence of nitrogen-fixing plants could lead to a significant drop in fertilizer demand, and consequently in both carbon emissions and nitrogen pollution. <br> <br>
<img src="https://static.igem.wiki/teams/5054/igemprojectdescriptionfigure1.png" alt='FEvolution of the nitroplast from the integration of the endosymbiotic UCYN-A bacteria into a eukaryotic.' width="400" height = "300"/>
<figcaption>Figure 3: Evolution of the nitroplast from the integration of the endosymbiotic UCYN-A bacteria into a eukaryotic <i>B. bigelowii</i> cell <a href="#cite14">[14]</a>.</figcaption>
</figure>
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<!-- <hr>
<h2>References</h2>
<ol>
<li id="cite1">Nitrogen - WUR.</li>
<li id="cite2">The nitrogen strategy and the transformation of the rural areas — Nature and biodiversity — Government.nl.</li>
<li id="cite3">National Oceanic US Department of Commerce and Atmospheric Administration. What is eutrophication?</li>
<li id="cite4">Jeff Elhai. Engineering of crop plants to facilitate bottom-up innovation: A possible role for broad host-range nitroplasts and neoplasts. 4 2023.</li>
<li id="cite5">Toename stikstofoverschot in landbouw door droge zomer 2022 — CBS.</li>
<li id="cite14">Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. Coale, T.H. and T. Nishimura. Nitrogen-fixing organelle in a marine alga. Science, 384:217–222, 2024. 6</li>
<li id="cite15">Angad P. Mehta, Lubica Supekova, Jian Hua Chen, Kersi Pestonjamasp, Paul Webster, Yeonjin Ko, Scott C. Henderson, Gerry McDermott, Frantisek Supek, and Peter G. Schultz. Engineering yeast endosymbionts as a step toward the evolution of mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 115(46):11796–11801, 11 2018.</li>
<li id="cite16">N.H. Nghia, I. Gyurj ́an, P. Stefanovits, GY. Paless, and I. Turt ́oczky. Uptake of Azotobacters by Somatic Fusion of Cell-wall Mutants of Chlamydomonas reinhardii. Biochemie und Physiologie der Pflanzen, 181(5):347–357, 1 1986.</li>
</ol>
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<h4>Bronze Medal Criterion #3</h4>
<p>Describe how and why you chose your iGEM project.</p>
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<p>Please see the <a href="https://competition.igem.org/judging/medals">2024 Medals Page</a> for more information.</p>
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<h2>What should this page contain?</h2>
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<ul>
<li>A clear and concise description of your project.</li>
<li>A detailed explanation of why your team chose to work on this particular project.</li>
<li>References and sources to document your research.</li>
<li>Use illustrations and other visual resources to explain your project.</li>
<p>We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be concise, accurate, and unambiguous in your achievements. Your Project Description should include more information than your project abstract.</p>
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<h2>References</h2>
<hr>
<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
