<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="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm"style="color: #C6EBE8;">[NitrogenWUR]</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>To combat global hunger and feed a growing population, an increase in global food production is crucial. This can be at least partially addressed through increasing crop yields, for which fertilizers are needed. Production of fertilizer is possible due to the Haber-Bosch process, 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><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #C6EBE8;">[Elhai2023 Engineering Neoplasts]</a>.</p>
<p>We expected, as the results of the experiment, that the light emitted by the primitive lux operon was a very faint blue light (similar to a schematic diagram of PV), which is very technological, but far from being called "LAMPS". Our project first set out to make our enzymes emit brighter and more colorful light for practical applications. We use both <b>protein engineering</b> and <b>metabolic engineering</b> to achieve the design goal of <b>increasing light intensity</b>.</p>
<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><ahref="https://www.cbs.nl/en-gb/news/2023/increase-in-agriculture-s-nitrogen-emissions"style="color: #C6EBE8;">[ToenameCBS]</a>. In addition to <strong>water pollution</strong> by leakage of nitrate, <strong>air pollution</strong> due to the conversion of nitrates 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><ahref="https://www.ipcc.ch/report/ar4/wg1/"style="color: #C6EBE8;">[AR4IPCC]</a>. For staple crops like cereals and maize, <strong>up to 40% of a farm’s operating cost is spent purchasing fertilizer</strong><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #C6EBE8;">[Elhai2023 Engineering Neoplasts]</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 <ahref="https://dutchnews.nl/news/2023/farmers-protests-in-the-netherlands/"style="color: #C6EBE8;">[ProtestingDutchNews.nl]</a>.</p>
<p>Altogether, there is a clear and urgent need for an alternative and environmentally-friendly solution to the nitrogen problem.</p>
<divclass="h"id="two"style="margin-top: -200px">
<divclass="h1">Problem</div>
<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="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm"style="color: #C6EBE8;">[NitrogenWUR]</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>To combat global hunger and feed a growing population, an increase in global food production is crucial. This can be at least partially addressed through increasing crop yields, for which fertilizers are needed. Production of fertilizer is possible due to the Haber-Bosch process, 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><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #C6EBE8;">[Elhai2023 Engineering Neoplasts]</a>.</p>
<p>We expected, as the results of the experiment, that the light emitted by the primitive lux operon was a very faint blue light (similar to a schematic diagram of PV), which is very technological, but far from being called "LAMPS". Our project first set out to make our enzymes emit brighter and more colorful light for practical applications. We use both <b>protein engineering</b> and <b>metabolic engineering</b> to achieve the design goal of <b>increasing light intensity</b>.</p>
<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><ahref="https://www.cbs.nl/en-gb/news/2023/increase-in-agriculture-s-nitrogen-emissions"style="color: #C6EBE8;">[ToenameCBS]</a>. In addition to <strong>water pollution</strong> by leakage of nitrate, <strong>air pollution</strong> due to the conversion of nitrates 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><ahref="https://www.ipcc.ch/report/ar4/wg1/"style="color: #C6EBE8;">[AR4IPCC]</a>. For staple crops like cereals and maize, <strong>up to 40% of a farm’s operating cost is spent purchasing fertilizer</strong><ahref="https://www.nature.com/articles/s41598-023-34214-3"style="color: #C6EBE8;">[Elhai2023 Engineering Neoplasts]</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 <ahref="https://dutchnews.nl/news/2023/farmers-protests-in-the-netherlands/"style="color: #C6EBE8;">[ProtestingDutchNews.nl]</a>.</p>
<p>Since the LuxCDABE system itself has a low brightness and is not enough to be used in real life, we decided to increase its brightness by some other methods. After reviewing the literature, we found that <b>when a fluorescent protein cp157Venus was fused to the C-terminal of the LuxB protein</b>, the brightness of the Lux operon system was significantly increased and the color of the light changed due to the <b>Biofluorescence Resonance Energy Transfer (BRET)</b> between LuxA, LuxB and Venus. So we constructed a plasmid containing the LuxB:Venus gene and expressed it. <sup>[1]</sup></p>
