iGEM-WrightState Deconta-Mn-ate:
Advancing Manganese Detection

Motivation

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 [1]. These processes can contribute to increased manganese levels in our groundwater, putting drinking water sources at risk [2]. In our home of south-west Ohio, the buried valley aquifer provides water for an estimated 2.3 million people [3], so this project is strongly connected to our community.

Manganese levels above World Health Organization limits are more likely to 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 recommended a provisional health based limit of just 0.08mg/L [4]. Given recent findings related to the negative health effects of chronic manganese exposure, we feel that now is an appropriate time to investigate methodologies for detection and remediation of manganese contamination.

Prior work

In 2022, our iGEM team designed a dual-plasmid system for the detection of manganese from drinking water (Figure 1). Our sensor utilized a dual-plasmid design which incorporated elements of the wild-type E. coli manganese homeostatic pathway. The sensor utilized a manganese-responsive gene promoter and riboswitch. In an effort to boost sensor performance, we used as second plasmid for the coordinated inducible expression of the manganese-binding transcription factor called mntR. With the dual plasmid system, we achieved detection down to 0.01mM manganese chloride (Figure 2, "uninduced dual"). However, the GFP output of our initial sensor design was low, limiting its potential utility in a fieldable device. Further, we found that increasing mntR levels completely blocked the function of our sensor (Figure 2, "induced dual"). Based on published literature regarding the E. coli manganese homeostatic pathway, we believe that this caused a downregulation of the manganese importer protein in response to increased levels of mntR [5].

Figure 1: Manganese detection strategy utilized by the 2022 WrightState iGEM team.

In whole-cell testing, the sensor alone (circles) showed a detection limit of around 0.1mM MnCl₂. When the second plasmid was added in the absence of IPTG (squares), the sensor showed improved performance, detecting MnCl₂ down to 0.01mM. Contrary to our expectations, when IPTG was added to induce higher level mntR expression, the induced dual system (triangles) did not respond to manganese. Based on the published literature, we feel that that inducing mntR resulted in a feedback downregulation of the manganese importer, thereby blocking sensor performance.

Figure 2: Dual-plasmid system (shown in Fig.1) detects 0.01mM MnCl2; IPTG induction blocks sensor function.

In whole-cell testing, the sensor alone (circles) showed a detection limit of around 0.1mM MnCl₂. When the second plasmid was added in the absence of IPTG (squares), the sensor showed improved performance, detecting MnCl₂ down to 0.01mM. Contrary to our expectations, when IPTG was added to induce higher level mntR expression, the induced dual system (triangles) did not respond to manganese. Based on the published literature, we feel that that inducing mntR resulted in a feedback downregulation of the manganese importer, thereby blocking sensor performance.

Project goals

Our 2023 iGEM project is built on the progress we made last year. In an effort to improve the sensitivity and fieldability of our sensor, we have pursued several strategies:

1. We sought to adapt our sensor to a cell-free platform to circumvent issues with manganese uptake experienced in our whole-cell assays.
• Using home-made cell-free lysate, our testing showed a GFP production response in concentrations as low as 0.01 mM MnCl₂ (0.5ppm).
2. We sought to improve alternative promoters and reporter proteins for use in our manganese sensor.
• We tested a T7-promoter driven sensor and sensors with deGFP and NanoLuc reporters. The NanoLuc sensor improved detection down to 0.007mM MnCl₂ (0.4ppm) and made it possible to image the response to manganese using a mobile phone.
3. We sought to make our manganese sensor more fieldable.
• We designed and tested a 3D printable luminescence imaging device (LID) to serve as a portable dark room. This hardware is designed to enable imaging of luminescence with a mobile phone and allow the end user to measure manganese levels outside of a lab setting. We also have a prototype mobile app in development to guide the user through imaging and analysis of manganese levels.
4. We sought to develop a strategy to chelate manganese from drinking water for use in conjunction with our manganese biosensor.
• We have developed a strategy for chelating manganese from drinking water in which we use E. coli to biomanufacture a tagged metal binding protein, bind this protein to silica beads, and use these in a column or filter format to purify water samples. Testing and implementation of this approach is planned as the next future step.

We hope that our system will provide a cost-effective and fieldable alternative to existing means of manganese detection and removal.

The idea for our project was built off of many of the values our team holds. We believe that all people have the right to clean water and therefore the right to know what contaminants are in their water. In many rural areas, water testing options are limited, leaving the residents to deal with the health effects [2]. Manganese is one major water contaminant, particularly in our home state of Ohio, where it can easily seep into sandstone aquifers. We also were inspired to work with manganese because little research has been completed on manganese bio-sensors such as ours. Manganese is a dangerous contaminant, but many people are not aware of the dangers or how to test for it. Our project will make manganese testing more accessible to those who need it most.

Citations