Integrated Human Practices

Background

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Context: When developing our project we were motivated by our environmental, moral, and scientific values. Environmentally, we know that high levels of manganese in water sources can be toxic to plants. In rivers and lakes, manganese contamination is usually caused by factory waste or fertilizer runoff; a method for sequestering manganese from water could combat this issue. Morally, we believe that everyone has the right to clean water, and therefore to know what's in their water. In rural areas, many people rely on private wells for their water; providing a simple manganese sensor could protect rural families. Scientifically, we found that the research into manganese as a water contaminant was less developed. The purpose of science is to explore the unknown and make it known; we worked to increase our collective understanding of manganese biosensors. These values contributed to the development of our project as well as how we aim to implement the sensor we have produced.

“In many small rural supplies, under resource limited circumstances, monitoring may be very limited. Whenever possible sampling should be designed to determine whether manganese is at concentrations in excess of the guideline value” [1].

Approach: The goals of our integrated human practices strategy were to:

1. Meet with experts in the cell-free field to learn about the benefits and limitations of the technique and assist in implementation.
2. Engage with experts in the water testing field to identify how our sensor would be used by the end user.
3. Meet with local researchers engaged in water testing to gain insight into the types of samples we (or the end user) would come in contact with during field testing of our sensor.
4. Work with engineering and design experts to identify what features an end user would need in a fieldable device and then implement that design in a 3D-printed piece of hardware.

With the help of the stakeholders in each of these areas, the team was able to design, build and test a robust NanoLuc manganese biosensor and 3-D printed Luminescence Imaging Device hardware to provide our end users with a fieldable manganese testing solution.

Stakeholders

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Summary of Interactions with Experts, End Users and Stakeholders

Dr. Nancy Kelley-Loughnane, Air Force Research Laboratories: Dr. Kelley-Loughnane provided us with information on cell-free transcription and translation systems, and recommended that we discuss Chushak et al 2021. She also initiated contact with Dr. Jorge Chavez Benavides and Dr. Kathryn Beabout. Dr. Kelley-Loughnane advised us to compare the performance of our sensor in both homemade and commercial systems because sensor performance is known to vary with different cell lysates.

• Contribution: Both myTXTL and homemade systems were tested. Based on this testing, we found that our sensor worked better with the homemade cell lysate.

Dr. Rajesh Naik, Chief Operating Officer, MINED XAI: During a team meeting, Dr. Naik shared his insights regarding utilizing and problem-solving cell-free systems. Dr. Naik has years of experience working with cell-free systems. We had faced a series of setbacks in the wet-lab, and asked for assistance in determining a path forward. He indicated that our cell-free tests of the GFP sensor showed a promising level of GFP protein, but the very low observed fluorescence suggested the GFP may be misfolded. He strongly recommended the use of a luciferase reporter because of the high sensitivity and bright output signal typical of luminescence assays.

• Contribution: Based on the recommendation of Dr. Naik, our team researched available luciferase reporter proteins, selected NanoLuc based on its superior luminescence output compared to other luciferase reporters, and ultimately integrated a NanoLuc sensor design into our project.

Dr. Jorge Chavez Benavides and Dr. Kathryn Beabout, Air Force Research Laboratories: Dr. Kelley-Loughnane introduced us to Drs. Chavez Benavides and Kathryn Beabout who met with us for a virtual discussion of cell-free systems. We presented the work we had done up to that point, and asked for their help in troubleshooting our cell-free approach. Dr. Kathryn Beabout shared many of the tips she learned working with cell-free systems. We discussed the lack of fluorescence in our early tests and the possibility that either Mn ²⁺ may be quenching fluorescence from the sfGFP reporter, or the addition of Cl ^- ions in the assay may be inhibiting the cell-free system. She advised that we set up our runs as kinetic experiments with reads every 10 minutes. She also suggested we swap our manganese induced promoter with a constitutively active promoter, and she offered plasmids for us to use. Dr. Beabout advised us on how to reduce evaporative loss in cell-free runs by adding water to adjacent wells in the microwell plate. Finally, Drs. Chavez Benavides and Kathryn Beabout offered to supply our team with cell-free lysate and the required buffers so that we could test our sensor in a homemade system.

• Contribution: We were able to test multiple sensor designs using the cell-lysate provided by Drs. Chavez Benavides and Kathryn Beabout. The material provided by their team allowed us to incorporate their suggested improvements and obtain an initial confirmation of sensor performance in a cell-free format. Further, it allowed us to test alternate promoters and reporters in our sensor design. This work would not have been possible without their support and guidance.

Miami Conservancy District (MCD): Our 2022 team initiated a collaboration with Mike Ekberg and MaryLynn Lodor of the MCD, an agency that provides water testing services for the Miami River Valley. Our 2023 team continued this collaboration. One of our team members accompanied Mr. Ekberg on a sample collection trip where they discussed sampling requirements and methodologies used in water sample collection and the effects of sampling location and depth on heavy metal levels in the water. The samples collected that day were shared with us so that we could compare manganese contamination measurements made using our sensor to those measured using standard methodologies.

• Contribution: Our conversations with Mr. Ekberg and Ms. Lodor provided us with a better understanding of the challenges facing private well owners and their needs as potential end users of our biosensor test. We used the samples they provided in the validation of our sensor and luminescence imaging device (LID) hardware.

Lake Campus Biology Department:Dr. Stephen Jacquemin supplied us with water samples collected from various sites around the Wright State Lake campus. He relayed information about his sampling strategy, testing of heavy metal levels from sediment in unfiltered water samples, and shared testing data of manganese levels typical in the watershed around campus.

• Contribution: Through our interactions with Dr. Jacquemin, we learned about the importance of sampling location and sample type when considering which heavy metals might be present. This information helped us better define the potential challenges an end user might face when using our sensor (e.g. excessive sediment, differences in pH and salinity). We used the water samples from Lake Campus to compare our sensor's performance to standard methodologies.

Andrew Kronenberger: Mr. Kronenberger assisted in the design of our luminescence imaging hardware. We discussed design elements that would improve ease of use, features of ergonomic design, and the potential use of recyclable resins for printing.

• Contribution: Multiple iterations of the hardware design were discussed, and his feedback contributed greatly to the overall design theme.

Wright State Engineering Department: Dr. Ahshan Mian provided critical insights into the materials needed for printing our luminescence imaging hardware, with a focus on ensuring proper printing and seamless integration of its components. We discussed available resins and their durabilitiy, printing quality and recylability.

• Contribution: We made several adjustments to the design to ensure that the sample holder could smoothly slide into the outer piece that holds the phone. He printed multiple versions of the hardware for us, and helped us to refine design tolerances and dimensions to improve the hardware.

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Acknowledgement to everyone who supported us: We want to express our gratitude to all the external sources that contributed their expertise and insights, guiding us on the (W)right path for the success of our project. The iGEM Wright State-OH team truly values the effort and thoughtful contributions that have played a vital role in moving our project towards success. We appreciate all the recommendations, data, and design contributions we received.

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