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 [LINK 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, YMT, H. pylori, P. aeruginosa, N. tabacum) and the recipient organism (Escherichia coli BL21-AI and Escherichia coli DH5a) 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 consume our cells or take up DNA lost from our cells. Unfortunately, we don’t know the answer to this question. Further studies would have to be conducted.

We have taken into account this risk assessment and have done our best to abide by these guidelines in our project.

Comprehensive Environmental Assessment (CEP)

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.

Altered Physiology:

Our modified E. coli cells differ from their E. coli BL21-AI and E. coli DH5a predecessors in that our modified strains contain the T7 promoter with a GST-YMT 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, the results of which are can be found here: https://2014.igem.org/Team:Cornell/project/wetlab/metallothionein . 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.

But I must explain to you how all this mistaken idea of denouncing pleasure and praising pain was born and I will give you a complete account of the system, and expound the actual teachings of the great explorer of the truth, the master-builder of human happiness.

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No one rejects, dislikes, or avoids pleasure itself, because it is pleasure, but because those who do not know how to pursue pleasure rationally encounter consequences that are extremely painful. Nor again is there anyone who loves or pursues or desires to obtain pain of itself, because it is pain, but because occasionally circumstances occur in which toil and pain can procure him some great pleasure. To take a trivial example, which of us ever undertakes laborious physical exercise, except to obtain some advantage from it? But who has any right to find fault with a man who chooses to enjoy a pleasure that has no annoying consequences, or one who avoids a pain that produces no resultant pleasure? On the other hand, we denounce with righteous indignation and dislike men who are so beguiled and demoralized by the charms of pleasure of the moment, so blinded by desire, that they cannot foresee.

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References

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