Team:RUM-UPRM/Poster

Poster: RUM-UPRM

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Mer-Nite to the Rescue

By: iGEM RUM-UPRM 2020




Abstract

For over six decades, Vieques, an island-municipality of Puerto Rico, was used as a military training site. Consequently, an array of contamination was left behind devastating the biodiversity and increasing health risks for the Viequenses. iGEM RUM-UPRM proposes Mer-Nite, a solution to decrease the contamination in Vieques. Phase 1 of the project consisted of designing a genetic system to absorb and bioremediate mercury through the expression of the mer operon, combined with a second circuit that will be activated upon the presence of RDX and degrade the contaminant through the expression of the xplA and xplB genes. Mer-Nite to the Rescue represents, not only, an alternative solution to decrease the contamination, but also, to conserve biodiversity, and improve the quality of life of Vieques.

Inspiration

In the 1940s, two-thirds of Vieques, an island-municipality of Puerto Rico, was used as a military training site by the U.S. Navy. During this period, the lands and waters of Vieques were used as bombing ranges and military practices, disturbing the Island. After years of on-going protests, the Navy finally abandoned Vieques in 2003 leaving behind an array of contamination; including heavy metals and organic compounds.These events led to Vieques being one of the most contaminated places in the Caribbean. This contamination was a major disturbance to the ecosystem and its biodiversity. In addition, Vieques presents a higher rate of cancer than mainland Puerto Rico.They current plans to clean up the area, has been to restrict access and include open air denotation which leaved behind toxic contaminants in the soils and waters of Vieques.

After completing our research and conversing with stakeholders, we chose to bioremediate Mercury and biodegrade RDX. Mercury affects mainly fishing since it tends to bioaccumulate in fish and crustaceans. When consumed, Mercury then can bioaccumulate in the consumers. RDX is a nitro explosive that is a known carcinogen to animals and humans.

Mercury

Detection




The first genetic device is activated by the presence of Mercury, which then activates Mercury-binding proteins that transports Hg (II) into the cytoplasm. Additionally, this device will induce an enzyme that will convert cell metabolites into acyl-homoserine lactones (AHL) which, in turn, initiates the second genetic device.

Bioremediation

This second device is responsible for the production of a lyase and reductase which will cleave the organic mercury molecules into its elemental non-toxic components, as well as reduce mercury into its gaseous state. The luciferase gene incorporated in this device is utilized as a reporter system to inform us of successful transcription of said device.

Killswitch

Finally, the last device is the kill switch, which is responsible for cellular lysis in the presence of blue light and the products of the second device.

RDX

Detection




The first device initiates transcription due to a stress response created by the presence of RDX. This device synthesizes AHL that will bind with the LuxR protein, activating the transcription of the second device.

Biodegredation

Upon activation, the second device will catalyze the reductive denitration and subsequent ring cleavage of RDX, followed by the expression of a green fluorescence protein functioning as a reporter gene.

Killswitch

Finally, the third device is activated by the end-products of RDX biodegradation, specifically nitrite and formaldehyde. This device will function as a modified AND Gate that will, eventually, initiate the bacterial lysis.

Modelling




To model our system and the reactions that take place within our devices, we have applied the Law of Mass Action kinetics to determine how the concentrations of the species change with respect to time. We consider a first order irreversible reaction as the following: From the first order reaction, we get the following differential equation which describes the change in concentration of the product with respect to time. For the differential equations used, a 17.1 amino acid/second value was used to calculate the translation rate for each gene based on their amino acid length.

For all three devices for each genetic device, we developed the ODE diagram representation in SimBiology and Concentration (mM) versus time (hr) plots to describe the system’s behavior. An overall analysis for all devices shows similar behaviors of the concentration as a function of time; the time is directly proportional to the concentration of translated proteins.

Mercury Device #2: Bioremediation of Mercury

RDX Device #2: Biodegredation of RDX

Hardware

We are also in the process of designing a bioreactor with the input of experts in the field to optimize the bioremediation and biodegradation. We are also taking into account biosafety concerns and making sure the genetically modified bacteria do not leave the environment.

Integrated Human Practices




By incorporating the ideas and thoughts of the public, we were capable of creating a two way conversation and being able to design and develop Mernite. For iGEM RUM-UPRM it is really important to grasp every aspect of our project. We are compromised with involving the community in the development of our proposed solution for Vieques. Hence why we decided to talk to stakeholders from different backgrounds which included professors, government faculty and researchers from the biological, social and engineering aspect. This way they all gave a new perspective and helped us on the creation of Mer-nite to the rescue.

Education

As part of our mission, iGEM RUM-UPRM wanted to educate and expand the synthetic biology knowledge in Puerto Rico. Due to safety concerns, we moved all of our activities to the Zoom ZOOM. We developed the SYNBIO 101: Summer Camp in which for a week high school students learned and experienced what is synthetic biology. The students created their own prototype, and presented it during the last of the summer camp. In addition, we promoted the Second Edition of the Synthetic Biology Week in which we discussed the diversity and the interdisciplinary aspect of synthetic biology. As a result, we educated over 300 Puerto Ricans and helped build a high school team from Puerto Rico that are preparing for the iGEM Giant Jamboree 2021.

Contribution

xplA/B (BBa_K3670000)




As part of our project, our team submitted the xplA/B biobrick to continue expanding the IGEM registry as well as to provide a RDX biodegradation part for future igemers. The xplA/B gene realizes the reductive denitration and subsequent ring cleavage of RDX.

Education

We educated the Puerto Rican public in a bidireccional way. First, infomed about synthetic biology and the problems we want to solve in Puerto Rico. Then the community helped us build on that knowledge, allowing us to further develop our project.

Phase 2

Acknowledgements

1. Dr. Patricia Ortiz Bermúdez & Dr. Carlos Ríos Velázquez, our PI’s advisors

2. Graduate student advisors Alejandro Mercado and Victor López

3. Government officials Eng. Vanessa Suárez & Mike Barandiaran from Wildlife Refuge

4. Agro. Sol Rosado Arroyo, President of PRABIA

5. Dr. Arturo Massol Deyá, Dr. Raquel Delgado Valentín, Dr. Rubén Diaz

6. Donations from Amgen and Goya were used for the SynBio 101 Summer Camp.

7.Revive and Restore made it possible for the registration of the team in the Giant Jamboree 2020.

References

[1] Chong CS, Sabir DK, Lorenz A, Bontemps C, Andeer P, Stahl DA, Strand SE, Rylott EL, Bruce NC. Analysis of the xplAB-containing gene cluster involved in the bacterial degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine.Appl Environ Microbiol. 2014 Nov;80(21):6601-10. doi: 10.1128/AEM.01818-14. Epub 2014 Aug 15. PMID: 25128343; PMCID: PMC4249041.

[2] Lee, B., Baek, H., & Oh, K. (2013). Use of an algD Promoter-Driven Expression System for the Degradation of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Pseudomonas sp. HK-6. Current Microbiology, 67(4), 480-486. doi:10.1007/s00284-013-0387-5 <

[3] Dávila-Santiago, L., DeLeón-Rodriguez, N., LaSanta-Pagán, K., Hatt, J. K., Kurt, Z., Massol-Deyá, A., & Konstantinidis, K. T. (n.d.). Microbial Diversity in a Military Impacted Lagoon (Vieques, Puerto Rico) as Revealed by Metagenomics. BioRxiv. doi:10.1101/389379

[4]Ortiz-Roque, C., & Lopez-Rivera, Y. (2004). Mercury contamination in reproductive age women in a Caribbean island: Vieques. J Epidemiol Community Health, 4(58), 756-757. doi:10.1136/jech.2003.019224

[5]Public health assessment Isla de Vieques Bombing Range, Vieques, PR. (2003) U.S. Department of Health and Human Services, Agency for Toxic Substances and Disease Registry. https://www.atsdr.cdc.gov/HAC/PHA/reports/isladevieques_02072003pr/index.html

[6] Massol-Deyá, A., Pérez, D., Pérez, E., Berrios, M., & Díaz, E. (2005). Trace Elements Analysis in Forage Samples from a US Navy Bombing Range (Vieques, Puerto Rico). International Journal of Environmental Research and Public Health, 2(2), 263-266.