Team:RUM-UPRM/Human Practices
Summary of Our Work
Our community's response towards our project and educational activities has determined that our work fits the criteria of being responsible and good for the world. After our trip to Vieques and being in contact with the several communities affected by the contamination present in the Anones lagoon, they were extremely willing to try out new solutions that involve cleaning the lagoon and reducing the expected cleaning time. Our project fits as one of those potential solutions due to its capacity to get rid of one of the most dangerous contaminants found in the Lagoon, while also providing safe and easy means like the use of a large-scale bioreactor to fulfill this role in an enclosed environment.
Likewise, the meetings we held with professionals in the areas of science, engineering, politics, among others, have given us good feedback and have given us their confidence to carry out our project. Last but not least, after having held our Summer Camp and our High School Intern Program, there has been an increased interest among younger students in using synthetic biology to solve local problems, which is our main goal. We have created awareness through these students, who are the future of our country and perhaps the continuity of our project. All these responses confirm that our project is reliable.
When designing our project we took into account the development of a way to reduce pollution in a sustainable way. In order to arrive at a more accurate solution, we were open to learning and making adjustments after every feedback from the public or from a professional. We were also honest with our community about what was happening inside and outside the laboratory and always tried to use simple concepts towards the public for a better understanding. This allowed us to create a truthful engagement with the public through our project.
Trip to Vieques
During the weekend of August 20-22, part of the iGEM-UPRM research group made a field visit to the Municipality of Vieques Island. Among the objectives of this visit, the aim was to visit different ecosystems on the Island, learn about the spaces impacted by military practices, interview people who lived during the events of the military occupation, and investigate the progress of the cleaning efforts of the impacted ecosystems. To meet the objectives, an academic tour was coordinated led by guides who, at the same time, are Viequenses residents, and have witnessed the before, during and after the military events of interest. The tour lasted about 4 hours, during which we were able to visit the permitted area of the Island and visit emblematic areas associated with military practices.
Some of the areas visited where the aircraft runway, the bunkers and the entrance, the main region stand out of main protests, conducting informal interviews with residents. From the moment we got on the bus, everyone we met provided us with historical and cultural information about the Island. Some of the story's told were about the mishandling of ammunition, the resistance that occurred during the time of occupation, criticism of the government and a distaste towards the local government for not accelerating the social development of the Island. In essence, all of them showed dissatisfaction with the current situation of the Island, remorse towards the Navy, thirst for social development, great cultural identity, and desire to close a chapter that was still open, unfinished or even forgotten.
Our first meeting was with 3 fishermen. Since the first approach there was a certain level of apathy. All three showed resistance to speak, as they initially referred us specifically to speaking with one of the three. They asked us specific questions like "what do you want to know?". Showing as if they were tired of repeating themselves. However, once we introduced ourselves and our purpose, they began to talk endlessly about the history of what happened with the Navy. It was to be expected, since we were precisely on the dock that was used by the Navy to transport weapons, soldiers and people associated with the practices, and the symbolism rooted in space was clear, and carried a great sense of struggle and resistance. In the conversation, the fishermen recalled the days of the bombing.
They also explained how these practices resulted in the restriction of access to much of the Island, and how the practices have delayed social development. According to them and historical facts, the lands were expropriated from Viequenses, which resulted in diminishing the space to build infrastructure, to develop tourism, and to compete with the other Lesser Antilles. In addition, the Navy bought or expropriated land that belonged to agricultural entrepreneurs of the cultivation of sugarcane, which also reduced to the end the agricultural agriculture of said monoculture.
Our second meeting was of a more didactic nature. While we were on our way to see the “magazines” that are certain types of bunkers that were used by the navy to store ammunition and other weapons, we ran into two historians familiar with the geopolitical issues of the Isla. Similarly, both provided information on the island, ranging from pre-Columbian to contemporary. Definitely, they denoted the presence of the navy as a decisive event in the history of the Viequenses, since they agreed that their passage on the island affected the social development of its inhabitants.
The third meeting and the one who had the most recent facts was of the second improvised tour that we had. This conversation was the most revealing about the current reality about the cleanup of the Vieques bombing. The driver, as well as a tour guide, is a worker at one of Fish and Wildlife's pump cleaning facilities. He is also closely acquainted with other types of information associated with other jobs. Regarding the employment contract, he told us that everyone must accept different health clauses. For example, all risks of contracting diseases associated with the different practices must be accepted, the risk of dying from them must be accepted, and, although we do not know the details of the contract, all these risks are offset by a large pay.
Within cleaning practices, he acknowledged that they focus mostly on visible white bombs. Those that are not visible, because they are covered by vegetation, are exposed after cutting down these spaces, or even burning them. The ones beneath the water are usually identified by local fishermen, divers, or others. Those that are underground, it is understood that they are not part of their immediate objectives. About his experiences, he told us that working in this place is peculiar, because the temperature is extremely high, unlike other places at a similar height.
Recopilating all this information, we were able to conclude that our project could aid in the cleanup of these areas. Also, that education is vital to understanding how contamination can affect and what can be done about it. The most important thing we learned is that viequenses are very proud of their island and of their culture and they will always search for the best of the interest of their people, welcoming every help that is presented to them.
Informal Interviews:
➜Fisherman 1 (Gabriel) 2 and 3: Gentlemen of approximately 60 years. Apparent colleagues in the fishing market. Born and raised in Vieques.
➜Historian 1: Male of approximately 60 years. Viequense with a great sense of history and belonging to the Island. Carpenter, music teacher, musical leader, and protester on the Island.
➜Historian 2: Male of approximately 50 years. Foreigner with a great sense of history and solidarity with the struggles against the Navy on the island. Carpenter.
➜Entrepreneur Ron Crab Caribbean: A young man from Vieques in his early 30s. They have a local family business that imports rum to the Big Island.
➜Tourist guide 1: Female of about 30 years. He has a daughter born and raised in Vieques. He studied something related to tourism.
➜Tour guide 2: Viequense male, 30 years old. He works in a polluted part of the marina as a janitor. He is also a driver and tour guide.
Take-Home Message
2. Only visible white bombs are cleaned, the toxic molecules in the air, plants, soils and so on do not seem to be of interest. (The detonations in the open air confirm this).
3. The Viequenses residents interviewed are eager to recover the contaminated lands, since they understand that this will catalyze social / economic development.
4. Recovering these spaces does not seem to be close. At the rate of cleanup efforts, its estimated it will take another 30 or 40 years… We think that is not a viable alternative to clean up molecular contamination. We understand that the ideal is bioremediation by bioaugmentation in the areas with the greatest contamination such as the Anones lagoon, and bioremediation by natural attenuation in the rest. However, molecules that cannot be catabolized and are toxic (such as heavy metals) appear to remain in space for a long time.
5. They are willing to try solutions that help reduce cleaning time.
Stakeholder Meetings
Meeting with Dr. Fausto
➜Background:
Dr. Fausto is a researcher that focuses on environmental microbiology and biotechnology. In which, he specializes in the microbial processes in the environment, especially degradation of contaminants. Currently, his research is to optimize the in situ removal of contaminants from the soil and groundwater in a low-cost, sustainable way, which is one of the reasons for contacting Dr. Fausto.
➜Adjustments/What we learned:
After giving him an overview of our project, Dr. Fausto provided some insight into our project, especially the design of our prototype. The discussion took direction into the incorporation of all the plasmids in one chassis or the option to divide the plasmid on multiple chassis. We took notes on this but felt it was a very ambitious thing to do because the successful transformation percentage could be reduced, so we decided it was not a feasible way. Another important point of discussion was the fact of characterization and quantifying each construct to validify the functions before experimenting with the interactions between the plasmids.
➜Next Steps:
A reporter gene was implemented for the first device, based on Dr. Fausto’s suggestions, not to just visualize the functionality but to have a manner to quantify the expression of these genes. We followed his advice and made all necessary changes so that the first device would have the reporter gene: mCherry. Eventually, each device would be quantified and characterized by their concentration and functions individually following similar implementations of reporter proteins.
Second Meeting with Dr. Fausto
➜Background:
Dr. Fausto is a researcher that focuses on environmental microbiology and biotechnology. He specializes in microbial processes found in the environment, more specifically, the degradation of contaminants. Currently, his research is to optimize the in situ removal of contaminants from the soil and groundwater in a low cost and sustainable way. One of the reasons for contacting Dr. Fausto, in addition to his research, was his expertise in primer design. Since our team was focusing on two cloning plans, one of these being Gibson assembly, we needed feedback on the design and parameters of our primers. Thanks to this interaction, we were able to learn the ideal parameters of primers used in Gibson assembly, as well as some personalized commentaries regarding Dr. Fausto’s experience in creating primers. Such comments helped us modify the parameters of the melting temperature, as well as take into consideration the length of primers. He also suggested that we try to join gene fragments in a single reaction, instead of doing it sequentially. Lastly, he emphasized the importance of doing a proof of concept for each device separately to ascertain its functionality. He specified that we needed to include a gene reporter in each device to facilitate and provide data fidelity between them.
➜Adjustments/What we learned:
In our second meeting, Dr. Fausto clarified the importance of the proof of concepts for each device, followed by testing the device with RDX in vitro to further validate the testing with the actual samples. In addition, some doubts of the application of the Gibson protocol were clarified. Especially the aspect of designing the complementary primers for our fragments.
➜Next Steps:
We will work on incorporating this procedure to validate the operation of the devices through the use of RDX in vitro. Also, apply Dr. Fausto´s recommendations regarding the creation of complementary primers and suggested platforms. In addition, we have to decide how to join the fragments (all together or by pieces), taking into account the information offered during the meeting.
Meeting with Dr. Carlos Santos
➜Background:
Dr. Carlos Santos Flores is a limnologist and a professor in the Department of Biology of the University of Puerto Rico in Mayaguez (UPRM). Since our sampling in Vieques would take place in the Anones Lagoon; therefore, Dr. Santos was contacted to gather expert opinions and recommendations towards methods of water sampling.
➜Adjustments/What we learned:
During the discussion, Dr. Santos emphasized the importance of taking into consideration what substances are in the lagoon, and at what time of the year the sampling will take place (salinity can increase or decrease depending on the season). He pointed out that the manual, “Standard Methods for Water and Wastewater Analysis” could be of further use for information regarding the sampling. One of the recommendations that he instructed us with was to at least take four samples, and at the end, he gave us encouraging words on the project and some other contacts that could be of help in our search for pre-sampling information.
➜Next Steps:
Thanks to the discussion with Dr. Carlos Santos we would continue our search for pre-sampling information. While searching we would keep in mind the valuable information that the professor emphasized, such as the system of the lagoon, other substances present at the lagoon, and the salinity during the different seasons.
Meeting with Dr. Jose Rodríguez Martínez
➜Background:
Dr. Jose Rodríguez Martínez is a principal investigator and assistant professor in the Department of Biology at the University of Puerto Rico in Río Piedras (UPRRP). He developed a technique to determine which nucleic acid sequences interact with any specific protein. The method is based on the use of nucleic acids called aptamers that interact with proteins to identify a signal, antibodies, presence of molecules, among others. The professor was contacted because of his extensive knowledge of detecting and identifying proteins and his opinion could evaluate the feasibility of our project.
➜Adjustments/What we learned:
The discussion with Dr. Rodriguez was an exchange of valuable information. Dr. Rodríguez presented us with questions to learn more about our project; we were able to answer some of them, and collect others to be taken into consideration for optimization and future implementation into our project. He pointed out that simulations were an important key point and offered to look into some of the simulations that we could use. We also discussed the possibilities of designing an RDX-sensitive protein with genetic engineering; nevertheless, we felt that was very ambitious, but would keep that precious information in mind.
➜Next Steps:
Thanks to Dr. Rodríguez Martínez we were able to further analyze our prototype, because of the questions raised by the professor. From the discussion with him, we came to an agreement that we would be sending him an outline that explains the prototype more deeply and he would look over it and raise more questions that could help us to further evaluate the feasibility of the genetic circuit and proteins used. In addition, he gave us key points regarding the Gibson Assembly technique and its importance in creating the most ideal primers.
Meeting with Dr. Nelson Cardona Martínez
➜Background:
Dr. Nelson Cardona Martínez is a professor at the University of Puerto Rico, experienced in student development and mentoring, Chemical Engineering, Science, and Catalysis. Dr. Cardona has an extended record of research and has a focus on the valorization of agricultural and forestry residues via the development of catalytic processes for the conversion of biomass-derived feedstocks into higher-value compounds. Dr. Cardona was contacted for his extensive knowledge of reactors. This meeting helped us understand how to design and operate our bioreactor in an effective way.
➜Adjustments/What we learned:
Dr. Cardona’s opinions and guidance towards making decisions regarding our bioreactor helped us identify new questions regarding its design and operability. Some of the things that were discussed were crucial information needed for the operation, such as concentration, mass transfer, and its behavior. Additionally, he discussed how the design parameters would depend on the concentration of the product that entered, and by these parameters, we would be able to know things, such as inflow and the purity of the out-product, which later would help us determine additional factors such as the quantity needed for consumption.
➜Next Steps:
We have a broader range of possibilities that will be helpful in the development of the bioreactor; therefore, further discussions on the design and operation of the bioreactor will be done taking into account the precious information that was discussed with Dr. Cardona.
Meeting with Dr. Lorenzo Saliceti
➜Background:
Dr. Lorenzo Saliceti is a chemical engineer at the University of Puerto Rico. His work explores bioreactor technology. The team contacted him for the possibility of collaborating throughout this cycle, since technical help was needed to build the bioreactor we intended to use to test our system on a larger scale.
➜Adjustments/What we learned:
A collaboration was agreed upon. We planned on having bi-weekly meetings during the summer and slowly build up the system. Dr. Saliceti wanted to give us a presentation on particle kinetics and another about introduction to bioreactors, for the whole team to learn the basics.
➜Next Steps:
Due to health concerns, we couldn’t collaborate during the summer, and progress on the bioreactor was hindered.
Meeting with Mike Barandiaran
➜Background:
In Puerto Rico, a large portion of Vieques is part of a National Wildlife Refuge, protected by the U.S. Fish and Wildlife (FWS). FWS is a government agency whose responsibility is the management of important natural resources; therefore, to do field sampling, we must follow proper procedures and permits. Mr. Mike Barandiaran is a representative of the FWS, and he was contacted for the discussion of steps to follow to coordinate a visit to Anones Lagoon.
➜Adjustments/What we learned:
During our discussion, Mr. Barandiaran provided details about the coordination of the visit. FWS and other authorities required us to prepare a formal proposal explaining the purpose, benefits, and protocols to be performed at the site. Mr. Barandiaran explained that we would be provided an escort from the US Navy and they would accompany us during the visit to the Anones Lagoon. Additionally, we discussed transportation logistics and directions for when we arrived at Vieques. Some recommendations were: traveling a day before sampling because of possible transportation difficulties and preparing different work plans with reduced team members (4, 6, or 8 team members) to ensure COVID-19 safety measures are taken.
➜Next Steps:
We will stay in contact with Mr. Barandiaran for updates about the final date for the visit to the sampling site. A work plan will be prepared beforehand, and once the agency gives us a confirmed date we could have an established plan to follow, including equipment, transportation, and lodging.
Meeting with Adrián González Costa
➜Background:
A priority in our list was the integration of the public and citizens of the Vieques municipality. To answer the problem, which would have been the best approach towards the public, we reached out and contacted senator Maria de Lourdes. Maria de Lourdes is an activist and has presented many environmental proposals, and one of them included working with the extraction of solid waste in Vieques. Mrs. Lourdes was represented by Mr. González Costa in the meeting and shared advice, opinion, and insight for our public approach to the viequenses.
➜Adjustments/What we learned:
Mr. González Costa told us that one of the most effective ways to approach the public at Vieques and get them to be aware of our intentions and project would be to contact persons involved in the Restoration Advisory Board (RAB) meetings. RAB are monthly boards done in Vieques in which information about the cleanup is exchanged among citizens, EPA, and the State.
➜Next Steps:
Contact to one or various members of the RAB board will be done so on following the advice of Mr. González Costa.
Meeting with Juan Dalmau
➜Background:
Juan Dalmau Ramirez is a Puerto Rican politician, attorney, a former senate member, and a former candidate for Governor of Puerto Rico. Mr. Dalmau has a broad range of experience in the topics of the military practice in Vieques and its social impact; therefore, to figure out the most effective approach to the viequenses, we contacted Dalmau.
➜Adjustments/What we learned:
The military practice done on the island of Vieques has created an immeasurable social impact on the viequenses community for a long time. This has left most viequenses with false promises of improving their quality of life from people whose motivation did not include helping Vieques. Regarding this, Mr. Dalmau gave us advice on how to effectively approach the community: with transparent information and with an academic purpose which is to be able, in some future, to improve environmental conditions through an alternative, non-invasive and non-damaging technique. He also gave us an opinion on how organizations in the community could be our most effective way of direct interaction with the viequenses; he emphasized: “community work must be achieved through collective integration to meet the needs of people, and thus gain the trust of the community, to be able to begin a joint process of collaboration.”
➜Next Steps:
Proper contact will be done with persons of the community. Furthermore, taking the advice of Mr. Dalmau we should get to know the community and be diplomatically prepared to explain any arising questions by the public.
Meeting with Dr. Sandra Maldonado
➜Background:
Dr. Sandra Maldonado is a professor at the University of Puerto Rico in the department of Biology, that focuses on mycology studies. The team contacted her and other mycologists because of the possibility of using fungal networks to transport our genetic material of interest. Since RDX is typically dispersed throughout the contaminated site, and on different levels of the soil, we thought this was a promising way to implement our project. To assess any chances of implementing this technology, we wanted to acquire more context we must include needing to analyze and thus met with Dr. Maldonado.
➜Adjustments/What we learned:
In the meeting, Dr. Maldonado made it clear that trying to opt for this route would prove difficult, both because of time constraints and financial needs. On top of that, if we wanted to implement this system effectively we needed to identify endemic species of fungi in the contaminated site, which at the moment was not possible because of the superfund site category of the area. She explained that once we had sampled and identified the fungal species, the next step would be to experimentally expose the strains to RDX and determine which was most resistant to the contaminant. After this, the next step would be to design and build a bioreactor to further get a sense of the studied system.
➜Next Steps:
After the meeting, we quickly understood that this would be hard for the team to accomplish, and discarded these alternatives as a proposed implementation.
Meeting with Dr. Carmen A. Vega Olivencia
➜Background:
Dr. Carmen Vega Olivencia is a Professor at the University of Puerto Rico in the chemistry department. She has broad experience in the techniques of bioremediation and along with other professors has published articles related to explosives and their effects on the environment. We contacted Dr. Vega Olivencia to broaden our possibilities for sampling our bacterial cultures quantitatively.
➜Adjustments/What we learned:
Dr. Vega Olivencia told us about Raman spectroscopy and cyclic voltammetry which could be two techniques and options to know the concentrations after exposing our transformed bacterias to the sample of water with RDX and contaminants. Raman spectroscopy and cyclic voltammetry are possible to test runs for more quantitative information on how the transformed bacteria with the devices would be responding to the samples.
➜Next Steps:
Thanks to Dr. Vega Olivencia, we got a further collection of information regarding sampling and contact information of personnel who could help mentor and perform the techniques. Necessary procedures and contacting will take place to take advantage of these potential methods of sampling our devices' performance. The following resources were provided to us by her during the conference:
Singh, Beer Pal, Arun Kumar, Armando P. Duarte, Segundo J. Rojas, Marielys Crespo-Medina, Hector I. Areizaga-Martinez, Carmen A. Vega-Olivencia, and M. S. Tomar. "Synthesis, characterization, and electrochemical response of iron oxide nanoparticles for sensing acetaminophen." Materials Research Express 3, no. 10 (2016): 106105.
Castellanos, Jorge, Carlos Rios-Velazquez, Freddie Morales, Viridis Miranda-Berrocales, José Liquet-Gonzalez, Isaac Cortez, Ricky Padilla, Carmen A. Vega-Olivencia, and Samuel P. Hernández-Rivera. "Cyclic voltammetry as a screening tool for the fungal degradation of 2, 4, 6-trinitrotoluene in aqueous media." International Journal of Environmental Analytical Chemistry 96, no. 10 (2016): 978-989.