Team:Cornell/testing/project/hprac/1

Introduction to CEA

As scientists, we are often inclined to reduce complex procedures down to simple, step-by-step protocols. Assessing risk and evaluating environmental impact are no exception; our first instinct this year was to create a universal checklist of regulations that every environmental iGEM project could fulfill in order to ensure environmental safety, the idea being that we could easily and systematically find answers to questions of environmental safety in scientific literature. However, upon speaking with Dr. Christina Powers, a biologist at the US Environmental Protection Agency, and further exploring the concerns relating to our own project, we decided to adopt a new approach to issues of human practices: Comprehensive Environmental Assessment (CEA).

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. The goal is to foster transparent discussion and use collective judgment to evaluate limitations and trade-offs in order to arrive at holistic conclusions about the primary issues that researchers should address in their research planning.

CEA & Synthetic Biology

While the Environmental Protection Agency primarily uses the CEA approach for nanomaterials, 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.

The Woodrow Wilson Center’s Synthetic Biology Project recommended that CEA be applied to more developed projects that were approaching field deployment in order to evaluate it as a risk-assessment approach for synthetic biology at large. This is where we come in: can CEA be successfully used to evaluate the risks of our field-deployable device? What are its limitations?

We began by attempting to apply the Synthetic Biology Project’s modified guidelines for prioritizing research questions to our own project as it currently stands.

Altered Physiology

The current versions of our genetically modified strains have a membrane protein in the Mtr pathway for anaerobic respiration knocked out and complemented on a plasmid. They differ from wild-type Shewanella oneidensis MR-1 in that their capacity for anaerobic respiration is upregulated in the presence of analyte (arsenic or naphthalene). This plasmid appears to be producing mtrB in a fully functional form that successfully completes the anaerobic respiration pathway when induced. One important difference is that when uninduced, the bacteria loses much of its anaerobic respiratory activity, and must rely significantly more on aerobic respiration; this is further discussed below.
In addition, our salicylate reporter strain contains the nah operon, which allows the cells to constitutively degrade naphthalene into salicylate. Salicylate, a metabolic intermediate in the Pseudomonas strain from which the nah operon was taken, is less toxic to the environment than naphthalene. P. putida G7 can further degrade salicylate into a catechol, an edible carbon source using the sal operon; this second operon is not present in our cells, so our Shewanella strains are unable to actively degrade salicylate. This means that our cells cannot use naphthalene as a carbon source, a conclusion that is supported by our naphthalene growth assays. The effects of salicylate production on the cells appear to be negligible, but this should be further explored in future characterization work.

One of our future plans is to integrate our constructs into the chromosomes of our strains, in order to ensure that mtrB is not saturating at its uninduced, basal expression levels. This poses new questions: will chromosomal integration alter the expression patterns of this engineered pathway? Will it interfere with other functions of the cells? Functionality can be altered depending on the position of the construct within the chromosome. This is our first forthcoming research priority: determining any potential adverse effects of chromosomal integration, carrying it out, and thoroughly testing the functionality of our new engineered strains.

Competition & Biodiversity

A second concern is that engineered strains could possibly outcompete wild-type species in the environment and pose a threat to biodiversity. Our first line of defense against this possibility is physical containment: the inlet and outlet of our current prototype are equipped with 0.2 µm filters, effectively eliminating the possibility that our engineered strains may interact with natural organisms. However, imagine that there could be a mechanical error: physical breakage of the device, slight discrepancies in filter pore sizes, et cetera, that could leave a slim possibility that our strains could come into contact with natural species. What then?
To answer this question, we return to the altered physiology of our strains: the primary distinguishing characteristic to note is that the cells cannot carry out the wild-type levels of anaerobic respiration unless they are induced. This means that in situations where oxygen availability is limiting, and toxins are not highly prevalent, our strains are less able to compete than wild-type Shewanella; this is our second line of defense. S. oneidensis is native to freshwater ecosystems in our area, so our strains should not be disruptive to biodiversity. In addition, we have observed that inserting the nah operon into cells seems to significantly increase their doubling time, so our salicylate reporter strains would be even less able to compete than our arsenic reporters.

However, what happens in the case that oxygen availability is not limiting? It is probable that our current engineered strains would be able to survive and thrive in aerobic conditions, and live alongside wild-type Shewanella. We need a third line of defense for this situation, a modification that would prevent our strains from being able to survive outside of our physical device. For this, we propose auxotrophy: in a truly field-deployable device, we would use a strain that had a gene for a key metabolite knocked out, so that the strain would rely upon supplementation of this nutrient in order to survive. We would then supplement this metabolite inside of the device, so that our strains would function properly within our device. If the bacteria were to somehow escape, their functionality would be severely impaired by the lack of nutritional supplementation, and they would be quickly outcompeted by wild-type strains.

This brings to the table a second research priority for field-deployability: engineer an auxotrophic strain of Shewanella to contain our constructs, and ensure that the likelihood of survival without nutritional supplementation is very, very low. We would also need to ensure, from a practical standpoint, that auxotrophy would not interfere with any other aspects of the physiology of the cells.

Evolutionary Prediction

Due to the relatively simple nature of our reporter system, we find it highly unlikely that our strains could evolve to possess any dangerous function. However, it is still possible that the promoter regions of our constructs could mutate and alter the expected patterns of promoter activity, or that the functionality of mtrB would be impaired by a deleterious mutation. The former possibility could serve to make the strain more genetically similar to the wild-type, and thus more able to compete; this presents us with another reason to pursue auxotrophy as a solution, basically in order to ensure that our strains are not alive for long enough for mutations to accumulate.

Gene Transfer Perhaps the most pressing concern with any synthetic biology project is the possibility of horizontal gene transfer from engineered to natural strains, and vice versa. Again, our first line of defense is physical containment, but barring that, what can we do to reduce the possibility that our strains would transfer unnatural functions to natural cells? The biggest shortcoming of our current system is that antibiotic resistance is used as a selective marker on our plasmids, thus allowing for the possibility that antibiotic resistance could be spread among natural populations via conjugation or passive transformation. While we could explore the possibility of using another form of auxotrophy as a selective marker on a plasmid, we believe that a more viable solution would be to eliminate the need for a selective pressure in the first place, namely by chromosomal integration. The transfer of chromosomal DNA is much less likely than the transfer of a plasmid, and, as mentioned above, chromosomal integration would solve other problems in the functionality of our reporters.

Limitations of CEA

CEA allowed us to think about crucial future work for our project in order to make it suitable for field-deployability. However, in our interactions with environmental groups, public officials in water quality management, and industrial groups, we encountered several important questions that were not built into existing CEA framework. Assessing the suitability of a synthetic biology-based device extends far beyond practical environmental risk assessment, into regulatory and economic concerns. The Presidential Commission for the Study of Bioethical Issues (PCSBI) released a report in December 2010 regarding important considerations for the ethics of synthetic biology projects, not all of which are encompassed by the CEA framework. In addition, as we approach field-deployability, the economics of the device become essential to assessing its viability as a solution to real-world water monitoring needs. These ideas are further explored in the remainder of this section.

References

http://www.nature.com/nature/journal/v483/n7387/full/483029a.html?WT.ec_id=NATURE-20120301 http://www.synbioproject.org/process/assets/files/6609/_draft/background_packet_for_cea_and_syn_bio_workshop_jul_28_2011.pdf http://www.synbioproject.org/process/assets/files/6609/_draft/final_cea_and_syn_bio_workshop_notes_wilson_center.pdf http://pubs.acs.org/doi/pdf/10.1021/es3023072