Overview

fishPHARM first started as an iGEM summer project motivated, but has quickly evolved into a novel start-up company with potential for immediate application and long-term impact. We have engaged in several entrepreneurship endeavors to facilitate the growth of fishPHARM as not only a synthetic biology based project, but as a locally-based business venture capable of curing BCWD worldwide. We have also partnered with a variety of fish hatcheries and research institutes in the New York State Finger Lakes Region to help take our discoveries from the lab bench and meet the demands of a growing consumer market. Through conversations with these hatcheries, which deal with bacterial coldwater disease daily, we are able to analyze the safety implications of deploying our product in the fish farming industry and compare the economic costs of our product to current solutions. fishPHARM is designed directly with the needs of your everyday fish farmer in mind. By collaborating with our consumers, we aim to develop a product that is not only safe and effective, but also is practical for treating BCWD in the real-world.

Hatchery Collaborations

We were inspired to start fishPHARM after seeing the devastating effects of BCWD in the local fish hatcheries of New York State. Fish hatcheries in the Finger Lakes region constantly deal with BCWD, and we sought to aid the local fish farmers in a more personal way through fishPHARM.

fishPHARM is unique because no iGEM team has ever shared a collaboration with fish hatcheries and fish research units. We have collaborated with Bath, NY Hatchery, Rome, NY Hatchery, and the Cornell Biological Field Station in order to take fishPHARM from the lab bench to real-world implementation. We have received a plethora of feedback from fish farmers that has driven us to redesign and improve upon fishPHARM in order to develop the most efficient mechanism to treat BCWD. Our collaborations are detailed below.

Bath, New York State Hatchery
Background: Bath Fish Hatchery, established in 1893, uses fish obtained from Cayuga Lake to produce roughly 86,000 pounds of rainbow, brown and lake trout annually [1]. The hatchery has had a tumultuous history, including a 1955 fire that leveled the main production facility, but remains an essential component of the New York aquaculture industry. The work the hatchery does ensures that the New York State Finger Lakes are teeming with fish during the annual angling season.
Collaboration: Cornell iGEM first toured Bath Hatchery over the summer, during which we were able to gain a behind the scenes look at how fish are bred and treated. Bob Sweet, a long-time employee at Bath Hatchery, spoke to us about how he had once seen BCWD kill up to 1000 fish/day at the hatchery. Bob also brought to our attention the lack of real-time fish tank monitoring systems at Bath Hatchery that could be used to prevent diseases like BCWD. This inspired the team to develop Heimdall such that fishPHARM could not only treat BCWD, but prevent it as well.

Economic Analysis

Oxytetracycline (branded as Terramycin) costs roughly $0.70/gram. Since the total amount of antibiotic required for a 10-day treatment is 25 g/100 lbs, treatment of a fish stock with Terramycin costs about $0.025 per dollar of fish [1]. However, Terramycin is already becoming obsolete as a treatment protocol due to the rapid formation of resistant strains [2]. While the antibiotic may appear cost effective, it cannot be relied upon as a long-term solution due to the rise of antibiotic resistance. Bacterial tolerance of chemotherapeutic agents could obligate fish farmers to use more potent antibiotics, which could have undesirable side effects on their stock. Furthermore, the evolutionary capability of Flavobacterium to overcome antibiotics suggests that reliance on such agents in the future is a questionable proposition.

Our tag consists of three core components: an on/off clamp, medical-grade tubing and 3-D printing material. Given that the clamps are $.23 each, the requisite tubing is $.21 a tag and the printing material is $1.40/tag, the total material cost per tag is $1.83 [cite]. This translates to about $.13 per dollar of fish. While this may seem like a nontrivial amount, fishPHARM guarantees users a peace of mind that Terramycin simply cannot match. fishPHARM’S EcnB peptide is biodegradable as well as, and thus does not pose the same environmental and health risks that antibiotics do.

It is clear that the negative consequences of Flavobacterium psychrophilum infection are acute. As reported by the New York Department of Environmental Conservation, an epizootic of BCWD once infected 25% of lake trout raised in New York State hatchery raceways [4]. Given the inedibility of infected salmonids, the organism has the potential to reduce the financial yield of a salmon population from $7/lb to $4.90/lb in this scenario. Since roughly 2.4 million tons of salmon are produced by aquaculturists each year, even a small incidence of BCWD would have dire economic ramifications.

Environmental Impact

Given the relatively benign nature of Entericidin B and its Escherichia coli chassis, the environmental consequences of fishPHARM are negligible compared to that of current mainstream BCWD treatment methods - namely antibiotics. While there is a slight risk of the Entercidin B disrupting the ecological microbiome present at its point of dissemination, the specificity of the toxin makes this unlikely.

EcnB is naturally found in the human gut genome and thus not a hazard to human health. The benign nature of the treatment protocol we have devised provides distinct advantages over oxytetracycline, an antibiotic used to treat BCWD, because abuse of such chemotherapeutic agents fosters resistance among targeted pathogens [5]. In fact, a study conducted between 1994 and 1998 among Danish trout farms recorded an oxytetracycline resistance rate of between 60 and 75 percent of Flavobacterium colonies sampled [6]. Bacterial resistance to agents such as amoxicillin, florfenicol and sulfonamides has also been reported, largely due to the organism’s ability to form extremely hardy biofilms [6]. FishPHARM thus constitutes an effective circumvention of the issue of environmental antibiotic resistance: by eschewing the use of any chemical agents, we prevent the formation of more dangerous pathogens.

Comprehensive Risk Assessment

Overview

It is tempting as scientists to think that we can treat risk assessment as we would treat any scientific protocols - that with a few key steps and critical considerations, we will always end up with the right answer. However, assessing risk, particularly for environmental projects, is not that simple. Thinking about potential impacts and risks often turns up more questions than answers, and it is difficult to know where to start. For this reason, we have employed three approaches to risk assessment. The first was developed by Cornell’s Environmental Health and Safety Department, pertaining specifically to work with recombinant organisms. The next was developed by the Environmental Protection Agency as a general environmental risk assessment and modified by both the Woodrow Wilson Center and our team for use on our synthetic biology project. Finally, we strived to embody the design principles set forth by the Presidential Commission for the Study of Bioethical Issues. Each approach has its limitations, but all of them have helped to inform our project design, research practices, and considerations for further development of our project.

Environmental Health and Safety (EHS)

Cornell’s Environmental Health and Safety Department lays the groundwork for determining safe research practices on campus, and greatly informed our own safety protocols. They specifically suggested the following risk assessment criteria for researchers working with recombinant organisms.

• Formation – The creation of a genetically-altered micro-organism through deliberate or accidental means. For our purposes, our modified organism was altered intentionally, thus we know all of the donor organisms and the recipient organism are not hazardous.
• Release – The deliberate release or accidental escape of some of these micro-organisms in the workplace and/or into the environment. Our product consists of naturally found peptides and not the genetically modified organism itself that essentially stays in the laboratory setting so the the surrounding environment will not have to deal with any release of micro-organisms.
• Proliferation/Competition/Establishment – The subsequent multiplication, genetic reconstruction, growth, transport, modification and die-off of these micro-organisms in the environment, including possible transfer of genetic material to other micro-organisms. The inclusion of the EcnB gene in our organism severely impedes growth, so even if our organism was to escape the lab into the environment, it couldn’t survive. Also, the EcnB gene has to be expressed so the organism wouldn’t be any different in that sense from other strains naturally found.
• Effect – The subsequent occurrence of human or ecological effects due to interaction of the organism with some host or environmental factor. Ideally, our cells would not have an effect on the environment or any other host as they are constrained to the laboratory setting. However, if there were to be a leak somewhere in the lab, the largest concern would be if another organism were to somehow take up DNA lost from our cells. This would require a naturally competent bacterial strain to come across a leak that yields an intact plasmid, and the plasmid would have to be able to replicate. In all likelihood, in the absence of selective pressure, the plasmid would actually be deleterious to the cell due to toxicity of the EcnB peptide.[1]

Comprehensive Environmental Assessment

The EPA’s Comprehensive Environmental Assessment (CEA) is a tool to allow scientists to broaden their perspectives by incorporating the experiences, expertise, and concerns of diverse stakeholders. CEA differs from traditional methods of risk assessment by recognizing that risk assessment is fundamentally a decision-making process in which scientists, experts, and the public should be engaged in transparent dialogue. The goal is to evaluate limitations and tradeoffs to arrive at holistic conclusions about the primary issues that researchers should be addressing in their research planning.

The Woodrow Wilson International Center for Scholars in Washington, D.C., recently launched efforts to lay out a framework to apply CEA to synthetic biology. This groundbreaking project set out to assess the CEA approach’s relevance to synthetic biology, in anticipation of the growing demand for synthetic biology-based solutions to global issues. They arrived at the conclusion that scientists should focus on some major areas of risk assessment: altered physiology, competition and biodiversity, evolutionary prediction/gene transfer. In the past, using this framework has helped to uncover its limitations and the ways in which we could improve our own approach to environmental risk assessment. [2,3,5]

Altered Physiology: We synthesized new proteins that are originally found in E. coli, but we induced them at a higher rate. The proteins are involved with osmoregulation, so the toxins would cause E. coli to lyse itself if the regulation somehow goes out of control. Thus, we found that the growth rate of our engineered cells was severely impaired due to the toxicity of EcnB.

Competition and Biodiversity: Since our cells never leave the laboratory environment and only the EcnB peptides enter the fish, the issue of competition is not as relevant as it would be for other projects. Our engineered plasmids cause our cells to be not as able to compete in the natural environment as the cells would grow too slowly and other bacteria would outcompete them. These new cells would not have time and resources to form their own niche.

Evolutionary Prediction/Gene Transfer: There is not likely to be a consequence when it comes to evolutionary prediction as there would be low evolutionary pressure for the bacteria to keep the plasmid in absence of the antibiotics. Furthermore, the cells with the engineered plasmids would be retained in the laboratory setting. Moreover, if EcnB gets more aggressive as a toxin, it would just lyse the cells before the culture has the chance to proliferate. The issue of gene transfer is not relevant and not likely since we are limited to and working in the laboratory setting when handling bacterial cells.

Bioethics

We designed our project in accordance with the ethical principles identified by the Presidential Commission for the Study of Bioethical Issues (2010). Our primary motive is to better stabilize our source of food by fighting a disease that is often harmful economically to communities and physically detrimental to many species of fish and the animals that rely on these fish as much as we do. We have also demonstrated responsible stewardship by considering the environmental implications of our project. The ecological impact of placing our genetically modified strain in water would be minimal because our filtration system will not allow bacteria to escape, and we have structured our future directions around risk management for the future. In addition, our project is an intellectually responsible pursuit: it cannot foreseeably be used to cause people harm. In the spirit of democratic deliberation, we launched our Humans & SynBio campaign, to get people thinking and talking about the ethics of synthetic biology. Our proposed system would be easy, cost-effective, and potentially usable on a global scale. Additionally, the modularity of our platform allows it to be adapted to the needs of different communities, in order to best serve global populations and environments.[4]

Limitations and Future Directions

We have learned from our studies that there needs to be more education about synthetic biology, as many people are not fully aware of this field. In addition, it would be helpful to have a comparison of opinions before and after we discuss what synthetic biology is. In order to make our human practices assessments more effective, we would need to have a broader sample size of people taking surveys and answering our questions. Because we live on a fairly liberal university campus with a constituency that socioeconomically slants towards the upper-middle class, our answers may be biased. However, if we were to interview a much larger and diverse sample size, our survey results would be more informative.

Risk assessment can constantly be improved upon. It would be interesting to know what versions of our project, within our portfolio of future ideas and applications, would be the most widely used and accepted. Could we use other materials/gels to make our FishBit more biodegradable? Would other industries be interested in using this tag system to treat other animals that face other diseases?

References

[1]Cornell Environmental Health and Safety. (2014). Biological Safety Levels 1 and 2 Written Program. Available from https://securepublish.ehs.cornell.edu:8499/LabSafety/biological-safety/biosafety-manuals/Biological_Safety_Levels_1_and_2_Manual.pdf

[2]Powers, C. M., Dana, G., Gillespie, P., Gwinn, M. R., Hendren, C. O., Long, T. C., Wang, A., Davis, J. M. (2012). Comprehensive Environmental Assessment: A Meta-Assessment Approach. Environ. Sci. Technol., 46, 9202−9208. http://dx.doi.org/10.1021/es3023072

[3]Presidential Commission for the Study of Bioethical Issues. (2010). New directions: The ethics of synthetic biology and emerging technologies. Washington, D.C.: PreDana, G. V., Kuiken, T., Rejeski, D., & Snow, A. A. (2012). Synthetic biology: Four steps to avoid a synthetic-biology disaster. Nature, 483. doi:10.1038/483029a

[4]Presidential Commission for the Study of Bioethical Issues.

[5]Synthetic Biology Project. (2011, July 28). Comprehensive Environmental Assessment and Its Application to Synthetic Biology Applications. Retrieved from http://www.synbioproject.org/events/archive/cea/