Description
In this page you will find details about the project's established problem and solution.
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Our Problem
Vieques is an island municipality of Puerto Rico that is known for its beautiful beaches and bioluminescent bay. During the 1940s, it was used as a military training site. For a span of 60 years, they tested live ammunition in two-thirds of the island, leaving multiple unexploded artillery in terrestrial and marine environments (Caro, 2008). They openly tested at an average of 180 days per year, being conducted eight miles away from 9,000 to 14,000 of Vieques civilians (Sanderson et al., 2017). This practice left behind an array of contamination of heavy metals and organic compounds. By 2005, the United States Environmental Protection Agency (EPA) listed the Vieques bombing range as a superfund site. One of the most contaminated bodies of water in Vieques is the Anones Lagoon. It is listed in the EPA’s Superfund National Priorities List indicating that the contamination poses a threat to human health and the environment (Davis et al., 2007). Moreover, the lagoon is directly connected to the Caribbean Sea, meaning the water’s current can carry the contaminants to open water.
The contamination left behind due to the military exercises has disrupted the ecosystem and biodiversity of Vieques. Species of flora and fauna have presented bioaccumulation of heavy metals affecting the local economy, which relies significantly on its fertile soils and fishing. In open environments, undesirable concentrations of pollutants could be dispersed through multiple biotic and abiotic processes. Contaminated soil with heavy metals has produced excessive levels of trace elements in edible plants cultivated in Vieques, suggesting the cause is due to the military exercises (Massol-Deyá, and Díaz, 2003). In addition, it also poses a threat to the marine ecosystem, particularly in the water and flesh of many living organisms nearby reef sites presenting the presence of explosive residues in these organisms (Porter et al., 2011). After the analysis of possibly hazardous chemicals on drinking water, two carcinogenic substances, hexahydro-1,3,5-trinitroso-1,3,5-triazine (RDX) and 2,4-DNT (DNT), were found in high concentrations in several freshwater sources at Vieques (Caro, 2008).
Currently, human health is at risk since the exposure to these precarious chemicals is not under control. The contamination has been identified in a site at unsafe levels, and it is reasonable to expect that some inhabitants may have been exposed to such contamination (Sanderson et al., 2017). To understand the distribution of the explosive residues to civilian exposure, Figure 1. illustrates the pathway from the explosion test area towards the inhabitants in the civilian area (Sanderson et al., 2017). In the aspect of the cleanup operations, the Navy focused on land-based high amounts of unexploded ordnance (UXO) removal and recently expanded to address UXO in freshwater, and marine environments, including the restriction of access to the contaminated lands, and removal of bombs by open-air denotation (McCaffrey, 2018). The main problem is the open-air detonation practice, resulting in the uncontrolled release of toxic contaminants to the environment, which only aggravates the problem of previously deposited contaminants within the environment.
Figure 1. Pathway from the explosion test area towards the inhabitants in the civilian area (obtained from Sanderson et al., 2017).
Our Solution
The iGEM-RUM 2022 team has continued to modify and optimize based on our last cycle’s project. The purpose of this genetic circuit, composed of three devices, is the biodegradation of RDX. Modifications were made to the first device to enhance RDX detection and provide more control to our module. The hmp::hcp fusion promoter activates in presence of RDX for the transcription of the luxI, luxR, and mCherry genes. The N-acyl homoserine lactone (AHL) produced by luxI will bind to the LuxR protein, subsequently activating our biodegradation module. Our first device also serves as a cell-signaling platform, otherwise known as quorum sensing, for regulation of the gene expression in response to fluctuation in cell-population density (Miller & Bassler, 2001). Once the bond of AHL-luxR protein is detected by the luxpR promoter, transcription of xplB, xplA and amilGFP genes is initiated. Their products will then create an RDX denitrification system which can take an aerobic or anaerobic pathway as shown in Figure 2. Subsequently, forming an RDX ring cleavage which is essential for the production of formaldehyde and nitrite. Once the detection and biodegradation module are transcribed our third device, the killswitch, will activate from the presence of the byproducts formaldehyde and nitrite. This module will produce colicin which will terminate neighboring bacteria by lysis to control biosafety. A diagram with the three devices working together is shown in Figure 3. We hope the modifications made on this cycle will help us get closer to the final construction of our genetic circuit and provide useful information to other teams regarding the elements of the biodegradation process.
Figure 2. RDX Aerobic and Anaerobic Denitrification Pathway.
Figure 3. SBOL representation of the RDX Genetic Circuit.
RDX will be biodegraded into formaldehyde and nitrite within a stirred-batch bioreactor. This bioreactor produces rotational movement to maintain the mixture homogenized, thus achieving the same concentration of reactants and products throughout the entirety of the vessel. Afterwards, the water with the reaction byproducts goes through a two-step denitrification and formaldehyde removal. The discharge flows from the first bioreactor to the second phase of the system. It consists of an ozone bubble diffuser for denitrification and an activated carbon filter for capturing the formaldehyde. Lastly, the water passes on to a third and final phase where aquatic plants will denitrify and remove formaldehyde as a second step to make sure none is left in the water.
Figure 4. Bioreactor System.
References
Caro, A. (2008). Diversity and Microbial Community Structure at a Former Military Ranges in Vieques (Puerto Rico). https://scholar.uprm.edu/handle/20.500.11801/967
Davis, J.S., Hayes-Conroy, J.S., & Jones, V.M. (2007). Military pollution and natural purity: Seeing nature and knowing contamination in Vieques, Puerto Rico. GeoJournal, 69, https://doi.org/10.1007/s10708-007-9095-7
Massol-Deyá, A., & Díaz, E. (2003). Trace Element Composition in Forage Samples from a Military Target Range, Three Agricultural Areas, and One Natural Area in Puerto Rico. Caribbean Journal of Science, 39 (2), 215–220.
McCaffrey, K. (2018). Environmental remediation and its discontents: The contested cleanup of Vieques, Puerto Rico. Journal of Political Ecology, 25(1). https://doi.org/10.2458/v25i1.22631
Miller, M. B., & Bassler, B. L. (2001). Quorum Sensing in Bacteria. Annual Review of Microbiology, 55(1), 165–199. https://doi.org/10.1146/annurev.micro.55.1.165
Porter, J. W., Barton, J. V., & Torres, C. (2011). Ecological, radiological, and toxicological effects of naval bombardment on the coral reefs of Isla de Vieques, Puerto Rico. NATO Science for Peace and Security Series C: Environmental Security, 65–122. https://doi.org/10.1007/978-94-007-1214-0_8
Sanderson, H., Fauser, P., Stauber, R.S., Christensen, J., Løfstrøm, P., & Becker, T. (2017). Civilian exposure to munitions-specific carcinogens and resulting cancer risks for civilians on the Puerto Rican island of Vieques following military exercises from 1947 to 1998, Global Security: Health, Science and Policy, 2:1, 40-61, DOI: 10.1080/23779497.2017.1369358
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