Project Description
Water is a necessity to sustain life. Yet, the unprecedented effects of climate change have triggered a hidden threat that could potentially devastate our aquatic environment — eutrophication. The issue of algae entering domestic and agricultural water supplies has sparked serious concern in the last 5 years in Korea. Currently, the Nakdong River, a river that has the longest span in the southern Korean peninsula, has seen record levels of toxic blue-green algae growth in the sections from which water is carried away into homes and fields. The river makes up 88% and 66% of Busan and Daegu’s water demands respectively (the second and third largest cities in Korea by population). Eutrophication is a yearly recurring issue in rivers and dam reservoirs that are used as major water sources, with the frequency and duration of algae occurrence increasing due to recent climate changes and physical environment changes (reduced precipitation, installation of weirs, etc.). While the growing trend of more frequent, more devastating cases of algae growth has been undeniable, it has only been in relatively recent years that the issue of eutrophication (if previously mostly regarded as an environmental issue) has entered the public safety conversation. Not only that, the rising questions of the origin of the local products produced near the Nakdong River will ultimately lead to low consumption impacting the local industry and lives of fishers, farmers, and individuals. In fact, there already is sufficient evidence of microcystin cross-contaminating rice produced from the downstream regions of the Nakdong River; in 2021, an average of 3.18µg of microcystins were found in every 1kg of rice. Estimating the daily intake of the average adult as 300g, translates to around 0.945µg of microcystins ingested every day. Also, the government allocated approximately 50 million dollars to eutrophication recovery, but the severe eutrophication in South Korea remains which indicates that a new monitoring system is worth being suggested.
To address this issue, our team decided to engineer a novel solution for early detection of eutrophication in lakes, streams, and rivers not only in South Korea, but around the world. Our team envisions a ‘surveillance system’ of cost-effective and robust nitrate biosensors that is capable of taking nitrate samples over a given body of water. The system will allow local authorities to detect hotspots and entry points of abundant nitrate and devise specific plans. Currently, the limited access to nitrate sensors restricts such a solution. Despite the unaffordability of accurate nitrate sensors, along with their incapability to cover vast regions, there has been a disappointing lack of attention in developing commercial, non-laboratory specific biosensors. Conversely, our team’s novel sensor using synthetic biology can identify the nitrate concentration underwater and express it on an easy chromatic scale — green, yellow, and red — depending on the degree of concentration. Our nitrate sensor incorporates the use of two reporter genes (GFP, RFP) along with the PyeaR promoter to allow the usage as an Arduino sensor, and the lon protease system to express different colors (Green → Yellow → Red) according to the nitrate concentration.
More specifically, our nitrate biosensor is designed to represent the color red under conditions in which nitrate exists, and green under normal conditions. To implement this, red was designed to allow the expression of mScarlet to be controlled by the yeaR promoter, as mentioned above, and green was designed to be controlled by mScarlet and polycistronic-arranged repressors (cI, MerR, EilR, CymR) and lon protease. Due to the inactivation of the yeaR promoter under normal conditions due to NsrR, the repressor and Ion protease are not expressed, causing mVenus to be expressed and maintained. Specifically, by introducing the previously known negative inducible expression system (pJEx-D/EilR (Ruegg et al., 2018), pCuO/CymR (Seo & Schmidt-Dannert, 2019), pL/cI (Valdez-Cruz et al., 2010), pHTH/MerR), the expression of mVenus is controlled by the negatively regulated promoter (pJEx-D, pCuO, pL, pHTH), expressing a green fluorescent protein at normal conditions due to not having a repressor. Still, when exposed to nitrogen, the yeaR promoter is activated to form the repressor and Lon protease along with mScarlet. As a result, the expression of mVenus is suppressed by the repressor, and the mVenus, which is fused with the lva tag at the c-terminal is quickly decomposed by the lon protease (Cameron & Collins, 2014). Therefore, our nitrate biosensor can easily measure the risk of nitrogen, which causes eutrophication, with the chromatic colors green and red. If the biosensor E. Coli designed in this way is coated on a film that combined alginate with han-ji film, a strip in which biosensors are very evenly distributed can be obtained. In addition, different colors can be obtained depending on the nitrate concentration by measuring the fluorescence expressed using laser and UV led. Since the color can be changed into electrical signals using the Arduino color sensor, it is possible to obtain a nonexisting nitrate sensor. Furthermore, as the nitrate concentration is measured by measuring two types of fluorescence intensity, more accurate measurement of nitrate concentration is possible.
While there still exists room for improvement, we truly believe that Monitro, being a biosensor, comes with numerous advantages. Monitro is created with a unique approach to developing an Arduino-compatible biosensor, which responds exclusively to nitrate ions. This quality allows for a more precise measurement of nitrate concentrations without the influence of other impurities within the body of water. Indeed, our sensor presents an outstanding precision, demonstrating sensitivity and accuracy in micromoles. We do not, however, plan to rest on our current successes. We aim to further utilize our technological innovation beyond the detection of nitrate, developing BOD (Biological Oxygen Demand) measuring sensors of various chemicals, such as chlorine and phosphorus, by applying a similar construction technique. By detecting nitrate concentrations through visual color signals, we envision Monitro serving as a visible solution to often invisible problems like eutrophication. We sincerely hope our contributions help inform society of environmental issues and expand the biosensor industry to form a real response.
