Contribution

Acknowledgement

Scientific discovery is built upon the work of those who come before us. Without the years of hard work and dedication contributed by scientists and researchers, our own project would not have been possible. Every study and paper adds to the knowledge of our species, and allows for future generations to see even further. Our team has contributed to the depth of knowledge in the iGEM registry by presenting a new composite part, and by sharing the struggles and successes we had with the parts we used. We hope that future teams will push the field further by building upon our work.

Our Contributions

When we began researching a potential project, we started by looking through the published literature on the health effects associated with manganese exposure and the methodologies for testing and remediation of manganese contamination. We were surprised to find a disparity in the research on the different water pollutants. Interestingly, we found a great depth of work on the detection of iron, mercury, cadmium, and copper contamination, but far less regarding manganese. We believed that by focusing our work at this under-researched area, we could improve scientific knowledge and provide a basis for future teams to do great work.
Throughout our research in 2023, the WrightState-OH team made significant contributions towards the bronze, silver and gold medal criteria (New Composite Part, Integrated Human Practices, and Hardware) by:

1. Developing a new composite part (BBa_K4681000)
Pertains to Gold Medal Criteria: Excellence in Syntetic Biology: New Composite Part

2. Developing methodologies and hardware to improve manganese detection
Pertains to Silver Medal Criteria: Engineering Success
Pertains to Gold Medal Criteria: Excellence in Synthetic Biology: Hardware

3. Promoting manganese awareness
Pertains to Gold Medal Criteria: Excellence in Synthetic Biology: Integrated Human Practices

4. Promoting STEM Education
Pertains to Gold Medal Criteria: Excellence in Synthetic Biology: Integrated Human Practices

Our contributions are detailed as follows:

Composite Part

Biosensor for luminescence-based detection of manganese contamination in drinking water

We have constructed and validated a biosensor for the detection of manganese in drinking water supplies which produces luminescence at a level sufficient for detection using a mobile phone camera. The mntP-riboswitch-NanoLuc (BBa_K4681000) is based on the mntP-riboswitch-sfGFP-double terminator composite part (BBa_K4217003) which was previously shown to produce a modest 2-fold increase in fluorescence with 1mM MnCl2 after 8 hours of incubation in a whole-cell assay format. The new composite part utilizes a NanoLuc reporter (BBa_K1159001) in place of sfGFP in a cell-free assay format which provides reduced testing time and a stronger output signal. This stronger output is sufficient for visualization and quantitation using a standard mobile phone camera.

Our testing of the mntP-riboswitch-NanoLuc-double terminator sensor in a pSB3K3 plasmid backbone showed the sensor responds to manganese in a dose-dependent manner (Figure 1A) and yields sufficient luminescence to be visualized and quantitated with a mobile phone (Figure 1B).

Figure 1: Performance validation of the mntP-riboswitch-NanoLuc sensor in a cell-free assay. (A) Luminescence generated by the sensor 2 hours after cell-free reactions were initiated. Error bars indicate standard deviation from 3 technical replicates, and dotted lines indicate 95% confidence intervals. (B) iPhone camera image of the resulting luminescence.

Hardware

Development of methodologies and hardware to improve manganese detection

Manganese testing of drinking water typically requires adherence to strict sample collection protocols followed by shipment to a local or regional testing laboratory. Groundwater sample collection generally requires either draining three times the volume of the well to get a clean sample (which is not always practical), or using a low-flow protocol which requires many parameters be met before the sample can be taken. Then the sample must be properly labeled and sent to the lab to ensure it is not mixed up with the hundreds of other samples the lab receives. Finally, after the sample is sent to the lab there is a waiting period before receiving the data for that sample. In our conversations with MCD workers, we found that labs in our region can take up to two months to provide results. A fieldable test that can be used on site where the water sample is collected would provide an immediate benefit to the user.

Our goal in designing a manganese biosensor for use at the point of sample collection was to deliver a test kit with the following attributes:

1. Methodology should be easy to follow. Having a protocol with an interactive guide, either in the form of a mobile application or quick reference protocol, is beneficial to the end user.



Our solution: To make our sensor user-friendly, we have developed a portable 3D-printed device to enable users to capture images of the sensor luminescence and a prototype mobile phone app to guide users through testing and analysis.

2. Assay time should be short. A test that can be performed in a couple of hours would be a dramatic improvement over having to send water samples out to a laboratory for testing.



Our solution: We have adapted our biosensor to a cell-free testing platform. The original manganese biosensor (2022 WrightState) was a culture-based test that required an overnight starter culture, dilution and growth to mig-log phase, and then 8 hours of assay time. Changing to a cell-free format removes all culture and growth time, and reduces assay time to around 2 hours.

3. No specialized equipment should be required. Many of the available test kits utilize specialized equipment. Less equipment means less room for error due to broken or missing parts and reduces the load taken into the field.



Our solution: Our new manganese biosensor design utilizes a NanoLuciferase (NanoLuc) reporter that generates sufficient luminescence for imaging and quantitation using a standard mobile phone. The NanoLuc sensor is described in detail in the parts page (BBa_K4681000). The luminescence imaging device is described in the associated Hardware page.

Awareness

Promoting awareness of water quality and manganese contamination

Background: Heavy metal contamination of drinking water supplies is a global issue, and extensive research has gone into developing strategies to Identify and get rid of heavy metals like iron, mercury, cadmium, and copper. However, research into manganese has lagged behind. Manganese is released into the environment through the natural erosion of various manganese-containing minerals, and through industrial processes including combustion of fossil fuels, application of fertilizers containing manganese sulfate (MnSO4), and as runoff waste in a wide range of manufacturing applications. These processes can contribute to increased manganese levels in our groundwater, putting drinking water sources at risk. Manganese levels above World Health Organization limits frequently occur in rural areas and developing countries where the water processing infrastructure is insufficient. With reports linking manganese contamination to neurologic dysfunction and Parkinson’s-like disease, the World Health Organization established a health-based value (HbV) of 0.4 mg/L. In 2021, the World Health Organization reduced this limit to just 0.08mg/L. Despite the health concerns associated with exposure to elevated manganese, available remediation methods used for removal or iron and arsenic, which are heavy metals that co-occur with manganese, poorly target manganese.

The higher levels of manganese in groundwater is especially damaging in areas which get drinking their water from aquifers. Our home state of Ohio gets most of its water from the large aquifers in the region, which opens us up to higher levels of manganese in drinking water. This can also impact rural communities which rely on well water, or communities without the infrastructure to test and treat their water. From these motivations we formed the goals of our project: to create a fieldable method of manganese detection and to increase awareness of manganese as a contaminant.

Contribution: We worked to spread information about the hazards of manganese contamination of drinking water through a series of local and web-based activities:

1. Discussions with private well owners:

In rural areas, where many people rely on drinking water from private wells, knowledge about the dangers of manganese and other contaminants is critically important. After the well is dug, an initial test is required by the EPA. However, after the first test, no further testing is required. Often private well owners go years or decades without testing their drinking water, although; testing might occur if someone in the household gets sick. Our project provides a method for rural families to test their well water for manganese contamination.
As a part of our education efforts, we worked with the Miami Conservancy District. MCD has hosted many Test Your Well events in the past, in which private well owners brought in water samples for free testing. We hoped to host another such event on the Wright State University campus and provide educational pamphlets and discussions about the dangers of manganese in drinking water. Unfortunately, no labs in the region were willing to take samples for such an event and we were unable to host a Test Your Well event. However, we were able to educate some private well owners closely connected with our team members about manganese contamination and our project.



2. Discussions with experts from local water testing companies:

Miami Conservancy District (MCD) - During our engagement with Mr. Ekberg of MCD, we had a discussion concerning our water testing project. He shared his experience from testing wells in the south-west region of Ohio. He also provided historical MCD data pertaining to the levels of contaminants found in groundwater samples from the 12 wells maintained by MCD. We were fortunate to obtain groundwater samples from him, a valuable contribution that significantly supports our research. Additionally, we explored the varying manganese levels and local water quality within the state of Ohio. Mr. Ekberg’s history of working with the Ohio EPA was also influential in our understanding of water quality. We learned not only what regulations are in place, but also how manganese is perceived in relation to other water contaminants by water quality experts in the region.




Wright State Lake Campus- In our work with Dr. Stephen Jacquemin, professor of Biology at Wright State Lake Campus, we discussed water testing methodologies which helped us understand the most effective techniques to get accurate and reliable results. We appreciate his insights and expertise in this area, and we are grateful for the water samples he provided from the Wright State Lake Campus. These samples made it possible for us to compare our sensor response with known manganese concentrations.




Environmental Health and Safety - We continued to discuss water testing methodologies with the Wright State University EHS department staff, highlighting the significance of precise and thorough testing protocols. Additionally, we researched the lengthy history of Wright State University water quality, which improved our contextual comprehension and allowed us to approach our project from a wider perspective.

Together, these interactions and contributions from various people and institutions have advanced our project and improved our understanding of Ohio's water quality history and testing procedures. We greatly appreciate everyone's willingness to meet with us and share their expertise. The connections we made within our community were essential in the progress and success of our sensor.



3. Discussions with the local scientific community:

We will be presenting information on our project and the health effects of manganese exposure from drinking water to our local scientific community in two campus events:

• Brown Bag Seminar to the Department of Biochemistry and Molecular Biology faculty, staff and students (October 24, 2023).
• Poster Presentation at the Wright State Celebration of Research to Wright State students and faculty (October 26, 2023).


4. Dissemination of information on the effects of manganese exposure:

We worked to increase the general knowledge of water quality and synthetic biology, as well as knowledge about the dangers of manganese as a specific contaminant. This work overlaps heavily with our general STEM education and will be further discussed below.

Education

Promoting STEM Education

Our team has been proactive in educating the community on water quality and treatment through a variety of outreach and education initiatives. While detailed on the Communication Wiki page, the contribution resulting from these efforts is highlighted here:

Spreading information about the field of synthetic biology: Our team has posted regular infographics called “Synbio Saturdays” on both Twitter and Instagram. These social media posts highlighted diverse topics, including our university's biochemical majors, synthetic biology's role in impossible foods, and various blotting techniques. These engaging snippets appealed to a broader audience, sparking interest in synthetic biology education. Such regular information shared in fun and interesting ways was an excellent way to educate people who would not normally be interested in learning about synthetic biology. By doing this work, we believe we have increased general awareness of synthetic biology and water quality science.

Presenting fun and educational programs to children and families in the community: The most influential way we educated those around us was by presenting fun and educational programs to children and families at the Boonshoft Museum of Discovery and the Center of Science and Industry (COSI). In collaboration with The Ohio State University iGEM team, we orchestrated engaging activity days at the Boonshoft in Dayton, Ohio, and COSI in Columbus, Ohio. We focused on introducing visitors to the captivating world of synthetic biology through orchestrating interactive displays. Our protocols brought science to life for more than 300 visitors. To educate visitors on issues related to water quality, we introduced the "Water Filter Engineering Design Challenge," and encouraged participants to engage in a hands-on activity that mimicked the critical process of water filtration. This activity shed light on how synthetic biology can be employed in engineering solutions to address real-world challenges, emphasizing the practical applications of the field. Additionally, we established the "Ask Scientist Sam" initiative, allowing visitors to interact with us and gain deeper insights into iGEM and various aspects of synthetic biology. By providing exercises for the children, we were able to share our excitement about science with them, and the parents could overhear the information we were sharing. Even though the event was not targeted at the parents, we shared many conversations with inquisitive adults about our research and iGEM as a whole. This interactive session enabled a dynamic exchange of ideas and facilitated a better understanding of the interdisciplinary nature and potential of synthetic biology.