<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="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm">[NitrogenWUR]</a> and chemical fertilizers <ahref="https://www.government.nl/topics/nitrogen-crisis">[TheGovernment.nl]</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="https://oceanservice.noaa.gov/facts/eutrophication.html">[US Department of Commerce What is Eutrophication]</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="https://www.wur.nl/en/newsarticle/Nitrogen-crisis-in-the-Netherlands.htm">[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">[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">[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/">[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">[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/">[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>
<p>However, since the brightness enhancement still fell short of our requirements and we wanted a richer color system, we screened and expressed brighter fluorescent proteins from FPbase <a href="https://www.fpbase.org" class="a-pagestyle">(www.fpbase.org)</a> that met the requirement of excitation wavelength.</p>
<p>However, since many fluorescent proteins only have sequence data but not fluorescence data, in order to identify whether their fluorescence performance is up to standard or not, we used <b>recurrent neural networks</b> to establish <b>a mapping model from sequence to function</b>, which helped us to select fluorescent proteins with incomplete information.</p>
<p>In the subsequent experiments, it was also proved that the model predicted more accurately. In addition, in order to break through the shackles of nature to create stronger fluorescent proteins, we used the <b>Generative Adversarial Network model</b> to generate sequences of fluorescent proteins that are not found in nature, which are predicted to have stronger brightness after simulation.</p>
<p>The fluorescence reaction substrate is normally increased by the introduction of LuxF and luxG proteins.</p>
<p>Additionally, the intensity of light is an essential criterium of LAMPS, leading us to involve two other parts: <b>LuxF and LuxG</b>. Previous study has shown that LuxF would <b>activate luciferases</b> (consists of LuxA and LuxB) by binding to the inhibitor of luciferases <sup>[2]</sup>. And LuxG also contributes to the luminescence with <b>extra substrates FMNH2</b> <sup>[3]</sup>. In a conclusion, in this section we combined basic lux operon, luxG and luxF to form luxCDABEGF and proved the composite part enabled E.coli to emit stronger luminescence than the ordinary lux operon, which is the basis of our whole design LAMPS.</p>
<divclass="h1">Make LAMPS more smart and reliable</div>
<p>In addition to better realizing the fluorescence function itself, we further explored the design of <b>synthetic biology</b> and <b>biocybernetics</b> in enhancing the application capabilities of engineering cyanobacteria.</p>
<p>We hope to make LAMPS "smart", capable of automatically turning on after entering the night and automatically turning off during the day without the need for manual control. Fortunately, cyanobacteria, as photoautotrophic organisms, have an inherent biological clock rhythm system. Through a series of protein interactions and phosphorylation modifications, it can generate oscillatory outputs in downstream promoter PkaiBC with a 24-hour cycle. <sup>[4,5]</sup></p>
<p>Connecting the lux luminescent gene cluster downstream of PKaiBC allows for diurnal and nocturnal rhythmic expression of the fluorescent genes.</p>
<p>As obligate photoautotrophic organisms, cyanobacteria's energy metabolism is largely self-sustaining, and it is particularly challenging to maintain the expression of a large amount of exogenous proteins at night. Therefore, adjusting metabolism to meet the fluorescence emission needs of LAMPS is important to considerate.</p>
<p>Metabolic optimization involves several aspects:</p>
<pstyle="margin-left:10px;text-indent:2em;font-size:min(1.3vw, 19px);"><spanstyle="color:#2B5D6F;"><b>1.</b></span> We expressed sRNA targeting Glgc, which is a gene encoding glucose-6-phosphate translocase. It reduces the expression of glucose-6-phosphate translocase, thereby decreasing the process of glucose-6-phosphate conversion into ADP-glucose and glycogen synthesis, allowing more material to flow into sucrose synthesis and increasing photosynthetic efficiency. <sup>[6]</sup></p>
<pstyle="margin-left:10px;text-indent:2em;margin-top:8px;font-size:min(1.3vw, 19px);"><spanstyle="color:#2B5D6F;"><b>2.</b></span> Additionally, we learned from the article <sup>[7]</sup> that cyanobacteria have a much higher concentration of pyruvate than acetyl-CoA, suggesting significant untapped metabolic potential. We plan to express pyruvate dehydrogenase to break down more pyruvate into acetyl-CoA, releasing this untapped metabolic potential and increasing product expression <sup>[7]</sup>.</p>
<p>Furthermore, considering biosafety, we designed a killing switch to prevent leaks. This is also a fully automated system composed of a logic circuit centered around a metal ion-responsive promoter. </p>
<p>We expressed nuclease A(nucA) under a constitutive promoter and linked its inhibitor gene, nuiA, downstream of a Ni ion-responsive promoter. When a certain concentration of Ni ions is added to the culture medium, it induces continuous expression of nuiA, allowing the microorganisms to survive. However, once the microorganisms leak and leave the culture medium, the concentration of Ni ions in the environment decreases, nucA is derepressed, and it cuts the DNA of cyanobacteria, leading to their death. <sup>[8]</sup> This design achieves automatic sterilization in the event of a leak and eliminates the need for manual handling.</p>
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<divclass="h1">Anticipate application scenarios and address climate issues</div>
<p>Considering the commercial feasibility and the value transformation of the technology, we worked with industrial design students to do some scenario exploration and product development with our technology.</p>
Reference: images from <aclass="a-pagestyle"href="https://www.etsy.com/listing/1310963748/35-38mm-fluorescent-stone-spherenight?epik=dj0yJnU9ODB3WGsxNUZnRVNOSkJaUEdXOXd0R3R1UTBUYXd1SlQmcD0wJm49NTMyb2ZyTThHZUdJQjVPdUt2cVcwdyZ0PUFBQUFBR1VZUEt3">here</a>
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<p>This product is suitable for atmospheric lighting in weddings/events/private villas. The wedding needs to decorate the outdoor lawn, you can use our natural degradation does not need to recycle the small luminous ball, a sprinkle on the ground, you can continue to glow for a few days, and do not need to recycle, the grass will be restored in a month (even more lush grass).</p>
<p>In the water park at night, people wear luminous bracelets, throw luminous volleyballs on the water, children's swimming rings are luminous, and luminous ducklings float on the water. This is also a scenario that can be realized with our technology.</p>
<p>The light-emitting ball could also be used as a marker for emergency relief. When firefighters enter the forest to look for missing tourists, they can drop the ball along the route up the mountain, so they can quickly mark the route.</p>
<p>In addition to the biodegradable balls, LAMPS can also produce products that can be used in a wide range of Settings: the roofs of public buildings such as stations/pavilions/corridors/flyovers, floor signs in parks/green Spaces/exhibitions, lighting in historic wooden buildings or waterfront areas, installation art, costume design or experimental art.</p>
<p>These two applications amplify the product benefits of biomaterials and combine the technical characteristics of LAMPS. This product makes our technology commercially viable and provides a promising value conversion path for this technology.</p>
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<divclass="h1">Reference</div>
<pstyle="text-align:left;">[1] Kaku T, Sugiura K, Entani T, Osabe K, Nagai T. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci Rep. 2021;11(1):14994. Published 2021 Jul 22. doi:10.1038/s41598-021-94551-4</p>
<pstyle="text-align:left;">[2] Brodl, Eveline et al. “The impact of LuxF on light intensity in bacterial bioluminescence.” Journal of photochemistry and photobiology. B, Biology vol. 207 (2020): 111881. doi:10.1016/j.jphotobiol.2020.111881</p>
<pstyle="text-align:left;">[3] Nijvipakul, Sarayut et al. “LuxG is a functioning flavin reductase for bacterial luminescence.” Journal of bacteriology vol. 190,5 (2008): 1531-8. doi:10.1128/JB.01660-07</p>
<pstyle="text-align:left;">[4] Snijder J, Axmann IM. The Kai-Protein Clock-Keeping Track of Cyanobacteria's Daily Life. Subcell Biochem. 2019;93:359-391. doi: 10.1007/978-3-030-28151-9_12. PMID: 31939158.</p>
<pstyle="text-align:left;">[5] Chavan AG, Swan JA, Heisler J, Sancar C, Ernst DC, Fang M, Palacios JG, Spangler RK, Bagshaw CR, Tripathi S, Crosby P, Golden SS, Partch CL, LiWang A. Reconstitution of an intact clock reveals mechanisms of circadian timekeeping. Science. 2021 Oct 8;374(6564):eabd4453. doi: 10.1126/science.abd4453. Epub 2021 Oct 8. PMID: 34618577; PMCID: PMC8686788.</p>
<pstyle="text-align:left;">[6] Li, S., Sun, T., Chen, L., and Zhang, W. (2021). Designing and Constructing Artificial Small RNAs for Gene Regulation and Carbon Flux Redirection in Photosynthetic Cyanobacteria. Methods Mol Biol 2290, 229-252.</p>
<pstyle="text-align:left;">[7] Hirokawa Y, Kubo T, Soma Y, Saruta F, Hanai T. Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942. Metab Eng. 2020 Jan;57:23-30. doi: 10.1016/j.ymben.2019.07.012. Epub 2019 Aug 1. PMID: 31377410.</p>
<pstyle="text-align:left;">[8] Čelešnik H, Tanšek A, Tahirović A, Vižintin A, Mustar J, Vidmar V, Dolinar M. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biol Open. 2016 Apr 15;5(4):519-28. doi: 10.1242/bio.017129. PMID: 27029902; PMCID: PMC4890671.</p>
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