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 & 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 & Safety (EHS)

Cornell’s Environmental Health & 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 (T7 bacteriophage, H. pylori, P. aeruginosa, N. tabacum) and the recipient organism (Escherichia coli BL21-AI and Escherichia coli DH5α) 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 filtration device includes a hollow filter reactor, which is specifically designed to hold cells inside, yet let water and other materials pass through it. The hollow fiber reactor is made of high flux polysulfone and has a molecular weight cut off at 5 kilodaltons, retaining about half of any molecule that is of that weight. It is highly unlikely that our cells would be capable of escaping the filter device.
• Proliferation/Competition – 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 metallothionein gene in our organism severely impedes growth, thus other cells in the environment will outcompete our genetically engineered strain.
• Establishment – The establishment of these micro-organisms within an ecosystem niche, including possible colonization of humans or other biota.
  Since our cells are both slow-growing and highly unlikely to escape from the filtration device, it is improbable that the organism will be able to create a niche and outcompete healthy cells within the ecosystem.
• Effect – The subsequent occurrence of human or ecological effects due to interaction of the organism with some host or environmental factor.
  Ideally, our project would not have an effect on the environment or any other host. However, if there were to be a leak somewhere in our system, 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 in our system 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 the increased metabolic load and would therefore probably be expelled.^[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 trade-offs 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 four major areas of risk assessment: altered physiology, competition and biodiversity, evolutionary prediction, and 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. Therefore, we have decided to incorporate a more in-depth cost-benefit analysis, information on existing water treatment practices, and public perspectives through our Humans & SynBio project.^[2,3,5]

• Altered Physiology: Our modified E. coli cells differ from their E. coli BL21-AI and E. coli DH5α predecessors in that our modified strains contain the T7 promoter with a GST-crs5 gene, which codes for Saccharomyces cerevisiae metallothionein, a metal-binding protein. Our E. coli have three different overexpressed transport proteins that work with the metallothioneins to uptake and sequester lead, mercury, and nickel heavy metal ions. We are using the lead transporter gene CPB4, originally from Nicotiana tabacum, under control by the Anderson promoter. The mercury sequestration system is composed of merT and merP, genes originally found in Pseudomonas aeruginosa. MerP is a periplasmic mercury ion scavenging protein. MerT is an integrated membrane protein that works to transport mercury ions into the cell’s cytoplasm. Finally, the nickel transporter is the nixA gene found in Helicobacter pylori.
  
  In addition to the three aforementioned strains, we constructed a fourth strain of E. coli, the reporter strain. We inserted amilCP behind both a nickel/cobalt activated promoter, Prcn, and a mercury activated promoter, PmerT. This functioned as a sign of when the above mentioned cells were metal saturated. Basically, when metal ions enter the reporter cell, the AmilCP is engaged, turning the cell blue, indicating that the other cells are saturated.
  
  Given these changes, we would expect that there would be a change in cell growth because the production of metallothioneins renders the strain slow-growing. We tested our theory through various growth assays, detailed in Metallothionein Results. We found that the growth rate of our engineered cells was severely impaired, such that over a period of one day, the total cell concentration was roughly half that of a wild-type cell.


• Competition and Biodiversity:
  In the extremely unlikely event of release from our device, our cells would likely be outcompeted very quickly by native environmental strains due to their decreased growth rate. Other cells would multiply much more quickly and overwhelm the engineered cells in most environments. However, in environments with exceptionally high metal concentrations, our engineered cells would actually have higher fitness than the wild-type cells due to their ability to sequester the metals (see Metallothionein Results). These conditions would presumably never be reached; it would take a massive quantity of concentrated metal solution, coupled with the physical destruction of our device, for this to ever be a matter of concern. The only other circumstance in which our cells would be expected to grow more rapidly than the wild-type would be under conditions of strong antibiotic selection. Our cells currently do contain antibiotic resistance genes, but further development of our strains could remove this by a well-designed chromosomal integration process.
  
  However, to avoid the possibility of release, we’ve implemented sturdy physical barriers between our cells and the environment. Within the filtration device, the genetically modified cells are held within a hollow fiber reactor, which seriously restricts the movement of particles above 20 kD, meaning that most individual proteins would be unable to escape, much less entire cells.


• Evolutionary Prediction:
  The only potentially dangerous component of our cells is once again antibiotic resistance, which would be eliminated in the development of a field-deployable product. A more difficult and therefore more interesting question is that of whether the cells would evolve away from their original function. The original induction of metallothionein production would saturate the cells with proteins, and in the absence of growth medium, the growth rate of the cells would be extremely slow. The proteins themselves would also be unable to escape from the reactor, so the total metallothionein concentration would in theory remain constant (barring degradation with time), even as cell concentration might very slowly increase. This then becomes an issue of timescale, and it seems that the bacterial cartridge would likely be replaced before this would become an issue.


• Gene Transfer:
  The issue of most concern would probably be the transfer of our antibiotic resistance genes from the E. coli to other organisms if the cells were to escape, but as mentioned earlier, this problem could be avoided entirely. It is also true that neither plasmid nor chromosomal DNA would be able to escape the fiber reactor, so engineered DNA would never have contact with the environment in the first place.


• Impact:
  This year Cornell iGEM surveyed a variety of people to get a better understanding of the general public’s opinion about Genetically Modified Organisms (GMOs) and the bioethics of the various applications. Not only did we create a survey and get hundreds of responses to pool data from, but we also did a general social networking project. Similar to Humans of New York, Humans & SynBio features individual interviews urging people to think deeper about synthetic biology to see their various opinions about it. Interestingly, we discovered that many people are unclear about the definition and purpose of synthetic biology. In addition, we noticed general hesitence towards acceptance of synthetic biology within food and animal products, but acceptance and curiosity about integrating synthetic biology in human life quality improvement. In our case, an overwhelming number of people thought that our project was an ethical use of synthetic biology. Albeit, it is important to consider the limitations of our survey, which are discussed later on.
  
  So how do people’s opinions about synthetic biology affect the risk assessment of our project? Well consider this: a project that people know very little about will generate fear. In our study, we found that a lot of people find genetically modified organisms to be a “risky” topic, but if we explained to them our project in more detail, they were more willing to accept it. Thus, there is a need for a broader education about synthetic biology to the public, and a need for transparent communication between scientists and the community.


• Cost-Benefit Analysis
  

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 public beneficence: to improve global environmental and public health by remediating metal contamination in water. 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, demonstrating justice and fairness in its intended implementation. 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. Too few people know about the field for there to be educated opinions about it. 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. What scale filter would be most effective? Which ones would be more efficient to produce and to market? Which ones would impact the most lives? The ideal implementation of our project will occur when the technological development is made to match the exact needs of the community.

References

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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. Dana, 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
3. 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
4. Presidential Commission for the Study of Bioethical Issues. (2010). New directions: The ethics of synthetic biology and emerging technologies. Washington, D.C.: 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/