Summary
The key to success is always moderation - especially when using nitrogen fertilizers in agriculture. Too little fertilizer will harm crop growth, yet excessive application will inadvertently cause ecological harm associated with nitrogen pollution.
Our proposed solution will work to reduce unnecessary fertilizer use while maintaining crop quality. To this end, we will develop an in vitro transcription-based biosensorr, “SAMURAI”, for sensing nitrate and ammonia in agricultural soils.
We aim to offer SAMURAI as a tool for every farmer to manage nitrogen levels in their soil, becoming a game changer in addressing nitrogen issues. This is the ultimate goal that ShowgNs strives for.
Explore how we can Show you the ground Nitrogen with our sensor!
Background
Nitrogen Pollution
The build-up of nitrogen pollution is a pressing global issue. These reactive nitrogen (Nr) are any nitrogen species apart from atmospheric di-nitrogen (N2). Ammonia and nitrogen oxides are particularly harmful to the environment: N2O has a greenhouse effect 265 times greater than CO2[1]. while ammonia released into bodies of water causes biodiversity loss due to eutrophication and damage to the fishing industry[2].
The nitrogen balance has historically been maintained through healthy natural circulation within the atmosphere, land, water, and biosphere. However, human activities, such as artificially fixing and utilizing nitrogen, introduce excessive amounts of reactive nitrogen into this cycle, disrupting its natural flow. This disruption compromises the health of nitrogen circulation.
Currently, the anthropogenic perturbation of global nitrogen cycles is well beyond the Planetary Boundary (resilience) of the Earth [3]. This problem is already in a catastrophic danger zone.
Cause：Agricultural Use
Currently, 85% of nitrogen pollution are attributed to the use of chemical fertilizers and other agricultural practices[4].
In the early 20th century, the development of the Haber-Bosch Process made possible the mass production and distribution of chemical fertilizers. A "Green Revolution" was achieved, greatly increasing grain yields and the global population we could support. On the other hand, over-introduction of nitrogen fertilizers has produced significant nitrogen pollution. This sentence is repetitive here.
However, the availability of fertilizer critically affect crop yields. With the world population exceeding 8 billion and food shortages once again becoming an issue, yields cannot be sacrificed.
To solve both of these problems, it is necessary to "optimize fertilizer application." . We want to accurately monitor the amount of nutrients required by the crop and apply the necessary amount of fertilizer at the necessary time.
Current Solutions and Their Problems
To optimize nitrogen fertilizer application, it is necessary to accurately understand the amount of nitrogen in the soil. Nitrogen in the soil comes and goes in multiple forms through complex reactions, but the majority of nitrogen is applied as ammonium. It is then nitrified to nitrate and is either absorbed by plants or leached out by rainfall. These two components have very different physicochemical properties and each must be measured separately for fertilizer optimization.
Currently, electrical conductivity (EC) is commonly used as a measure of soil nitrogen. EC values are positively correlated with nitrate concentrations, but the gradient of the correlation varies widely depending on the nature of the farmland.It is also not suitable for determining accurate nitrate concentrations because other nutrients are also included in the measurement[5].Electrode-type nitrate sensors have also been developed, but they are affected by disturbing ions in the soil and are also expensive.
Devices capable of measuring ammonium ion concentrations in soil itself are not widely available.
In conclusion, optimal fertilizer management is not possible with existing devices.
Our Project
Inspiration
One member of our team had been interested in soils and nitrogen in soils since elementary school. So when we researched topics related to soils and nitrogen, we got interested in the nitrogen issue. And we found that one of the major sources of nitrogen leaching was agricultural land. Furthermore, since many of our members major in agriculture, we decided to start a project to address nitrogen runoff from agricultural lands.
Our Goal
Our project is to develop an in vitro transcription-based biosensor “SAMURAI” that can affordably detect concentrations of ammonium and nitrate ions in real-time.
In our system, the concentrations of ammonium and nitrate ions are converted into the transcriptional activity of T7 RNAP by using a variety of proteins. The concentration of transcribed RNA then becomes observable with either luminescence, fluorescence, or dye outputs. Our goal is to help farmers manage fertilizer application based on these quantified results.
We adopted an in vitro transcription-based approach while taking advantage of the high specificity of the enzyme. This approach gave our biosensor features such as ease of handling, rapid detection, prominent visibility, affordability, and compliance with the Cartagena Act.
The input part of our sensor is designed based on prior research called ROSALIND [6]. ROSALIND is a biosensor using a transcription factor that dissociates from DNA as the ligand concentration increases. When bound to DNA, transcription by T7 RNAP is inhibited. As the ligand concentration increases, the transcription factor dissociates from the DNA, and transcription is activated. We apply this mechanism directly to the TnrA system, which will be explained later, and for proteins that interact with ligands in ways that ROSALIND cannot accommodate, we propose mechanisms using Leucine Zipper proteins or Split T7 RNAP. Regarding the output, in addition to trends, we propose systems using luminescence via luciferase and color changes induced by Cas7-11. The Cas7-11 system was inspired by the project of iGEM McGill 2023, Proteus [7].
Gene Circuit
Our genetic circuit system is composed of three systems: ammonium sensing, nitrate sensing, and reporting.
1. Ammonium Sensing system
1-A GlnR
GlnR is an ammonium-dependent transcription factor and GlnA (GS) is a glutamine synthase in microorganisms such as Bacillus subtilis. GlnA synthesizes glutamine from glutamate, ammonium ion and ATP. GlnA is feedback inhibited by glutamine, leading to the formation of GS-FBI. This complex stabilizes GlnR and its binding to DNA, thereby ensuring that GlnR remains attached to the DNA [8].
In this project, we designed a protein that fuses GlnR with leucine zipper A3.5, and another protein that fuses T7 RNA polymerase (T7 RNAP) with leucine zipper B3.5 [9]. By using a mutant T7 promoter with low affinity to T7 RNAP based on previous studies, we designed a system in which T7 RNAP can initiate transcription only when GlnR is bound to DNA [10].
1-B TnrA
TnrA is a member of the transcription regulator family that controls nitrogen metabolism in B.subtilis, which also includes GlnR [8]. Contrary to GlnR, TnrA binds to DNA only when GlnA-FBI is not present. Therefore, it can bind to DNA only when ammonium ion levels are low and inhibit transcription of T7 RNAP.
2 Nitrate Sensing system
2-A NasR
NasR is a protein that gains binding activity to specific RNAs based on nitrate concentration [11]. We utilized split T7 RNAP, with one part fused to NasR and the other part fused to the RNA-binding protein MS2. In the presence of nitrate, the complex formed by the NasR fusion protein binds to RNA, leading to the reassembly of T7 RNAP and subsequent induction of transcription [12].
2-B NLP7
NLP7 is a protein involved in nitrate metabolism derived from Arabidopsis thaliana, which undergoes structural changes in response to nitrate concentrations. Due to its significant structural changes depending on nitrate levels, a sensor using Split-mCitrine has been developed in previous research [13]. We have designed a genetic circuit where structural changes in response to nitrate concentration reassembly Split-T7 RNAP, thereby activating transcription.
3.Reporter System
3-A luminescence by MS2/PP7-Nanoluc
MS2 Coat Protein and PP7 Coat Protein are proteins that selectively bind to RNA with high affinity [12]. There is prior work showing that it is possible to reassemble split luciferase on RNA using fragments of split luciferase fused to MS2 and PP7 [14]. We will apply these results to construct a system in which luminescence occurs in the presence of ammonium and nitrate ions.
3-B fluorescence by Broccoli2x
Broccoli2x is a fluorescent aptamer that fluoresces when bound to an organic molecule called DFHBI-1T and is used to detect transcription reactions[15]. Because fluorescence is highly quantitative, proof of concept in the laboratory is done primarily with Broccoli 2x.
3-C color change by Cas7-11/chromoprorteon
Cas7-11 is a protein that activates csx29, an endopeptidase, when it binds to target RNA. Activated csx29 cleaves specific sequences of csx30[16]. Inspired by a previous study in which fluorescent proteins were released into solution by TEV Protease, we decided to create a mechanism to release dye proteins into solution in response to RNA[17].
To balance compliance with biosafety and the widespread use of biosensing technology, it is essential to develop substance-sensing systems using cell-free systems, as mentioned above. Therefore, to enable future applications for sensing various substances, we have platformized the sensing system used in this project and named it MITSUNARI (Modular In vitro Transcription-based Sensing platform Utilizing Notable outputs And protein-molecular Interaction). Theoretically, MITSUNARI can be applied to any protein that undergoes substrate-dependent DNA binding, RNA binding, protein interaction, or protein structural changes.
For detailed explanations of the genetic circuits, please refer to the Design page, and for a detailed description of the platform, see the Parts page.
Hardware
To accelerate the social implementation of SAMURAI, it is desirable to have hardware that can be installed in farmlands and automatically handle sampling and measurement. Therefore, we have developed a device that automatically collects soil, extracts it into a solution, mixes it with the cell-free censoring system, and conducts the measurement. For a detailed explanation of the hardware, see the Hardware page.
Entrepreneurship
A business perspective is essential for the widespread adoption of SAMURAI. Furthermore, since the main objective of this project is to solve environmental issues, it presents challenges that differ from regular businesses. Therefore, we have created a business plan to prepare for future societal implementation with advice from experts. For more details on entrepreneurship, see the Entrepreneurship page.
Reference
[1]Climate Change 2014 Synthesis Report. landportal.org. Published July 21, 2017. Accessed June 15, 2024. https://landportal.org/fr/library/resources/978-92-9169-143-2/climate-change-2014-synthesis-report
[2]SCIENCE BRIEFS on TARGETS, GOALS and MONITORING in SUPPORT of the POST-2020 GLOBAL BIODIVERSITY FRAMEWORK NEGOTIATIONS. Convention on Biological Diversity; 2022. Accessed June 16, 2024. https://www.cbd.int/doc/c/c874/6eb7/813f0201cd67299c9eb10a4a/wg2020-04-inf-02-rev-02-en.pdf
[3]Richardson K, Steffen W, Lucht W, et al. Earth beyond Six of Nine Planetary Boundaries. Science Advances. 2023;9(37). doi:https://doi.org/10.1126/sciadv.adh2458
[4]Campbell BM, Beare DJ, Bennett EM, et al. Agriculture production as a major driver of the Earth system exceeding planetary boundaries. Ecology and Society. 2017;22(4). https://www.jstor.org/stable/26798991
[5]Vyavahare GD, Lee Y, Seok YJ, Kim HN, Sung J, Park JH. Monitoring of Soil Nutrient Levels by an EC Sensor during Spring Onion (Allium fistulosum) Cultivation under Different Fertilizer Treatments. Agronomy. 2023;13(8):2156. doi:https://doi.org/10.3390/agronomy13082156
[6]Jung JK, Alam KK, Verosloff MS, et al. Cell-free biosensors for rapid detection of water contaminants. Nature Biotechnology. 2020;38(12):1451-1459. doi:https://doi.org/10.1038/s41587-020-0571-7
[7]igem.wiki. https://2023.igem.wiki/mcgill/
[8]Wray LV, Fisher SH. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase. Molecular Microbiology. 2008;68(2):277-285. doi:https://doi.org/10.1111/j.1365-2958.2008.06162.x
[9]Thomas F, Boyle AL, Burton AJ, Woolfson DN. A Set of de Novo Designed Parallel Heterodimeric Coiled Coils with Quantified Dissociation Constants in the Micromolar to Sub-nanomolar Regime. Journal of the American Chemical Society. 2013;135(13):5161-5166. doi:https://doi.org/10.1021/ja312310g
[10]Hussey BJ, McMillen DR. Programmable T7-based synthetic transcription factors. Nucleic Acids Research. 2018;46(18):9842-9854. doi:https://doi.org/10.1093/nar/gky785
[11]Chai W, Stewart V. NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca m5al nasF operon leader in Vitro. Journal of Molecular Biology. 1998;283(2):339-351. doi:https://doi.org/10.1006/jmbi.1998.2105
[12]Braselmann E, Rathbun C, Richards EM, Palmer AE. Illuminating RNA Biology: Tools for Imaging RNA in Live Mammalian Cells. Cell Chemical Biology. 2020;27(8):891-903. doi:https://doi.org/10.1016/j.chembiol.2020.06.010
[13]Liu KH, Liu M, Lin Z, et al. NIN-like protein 7 transcription factor is a plant nitrate sensor. Science. 2022;377(6613):1419-1425. doi:https://doi.org/10.1126/science.add1104
[14]Ng KK, Cole KH, Halbers LP, et al. A modular platform for bioluminescent RNA tracking. bioRxiv (Cold Spring Harbor Laboratory). Published online July 3, 2022. doi:https://doi.org/10.1101/2022.07.02.498144
[15]Kartje ZJ, Janis HI, Mukhopadhyay S, Gagnon KT. Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter. Journal of Biological Chemistry. 2021;296:100175. doi:https://doi.org/10.1074/jbc.ra120.014553
[16]Strecker J, Demircioglu FE, Li D, et al. RNA-activated protein cleavage with a CRISPR-associated endopeptidase. Science. 2022;378(6622):874-881. doi:https://doi.org/10.1126/science.add7450
[17]Yu X, Sun J, Wang W, et al. Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose. Bioprocess and biosystems engineering. 2017;40(7):1101-1110. doi:https://doi.org/10.1007/s00449-017-1772-4