<divclass="h1">Introduction: Mission, Problem and Solution</div>
<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 <ahref="#cite1"style="color: #185A4F;">[1]</a> and chemical fertilizers <ahref="#cite2"style="color: #185A4F;">[2]</a>.</p>
<p>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 <ahref="#cite3"style="color: #185A4F;">[3]</a> that negatively impacts the local biodiversity. This highlights the need of the hour: <strong>tackle the nitrogen crisis without negatively affecting food production</strong>, which still depends highly on fertilizers.</p>
<p>The Nitrogen Action Programme, introduced by the Dutch government in 2015, aimed to reduce nitrogen deposition, particularly in agriculture due to fertilizer use and ammonia emissions. However, in 2019, the Council of State deemed the programme <strong>insufficient</strong>, highlighting that nitrogen emissions were not just affecting rural ecosystems but also impacting urban development. As a result, new residential construction projects were halted until nitrogen emissions could be adequately compensated for, exacerbating the already critical housing shortage in the Netherlands <ahref="#cite1"style="color: #185A4F;">[1]</a>. This demonstrates how agricultural nitrogen management has far-reaching effects beyond the environment, directly influencing urban issues like the housing crisis, thereby emphasizing the urgency of addressing both challenges in tandem.</p>
<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>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>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>
<divclass="h2">Would We Truly Eliminate the Need for Fertilizers?</div>
<divclass="h2">How Much Money Would We Save?</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>
<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>
