Team:UC Davis/Design
CenozoicTaking a step back, looking at the biotechnology field as a whole, many examples readily present themselves, in which products or services were offered or presented in ways which were incompatible with maintaining public trust. Among these are cases of pharmaceutical executives arbitrarily increasing the prices of lifesaving therapeutics by factors of several thousand percent, or the infamously unpopular patents of Genetic Use Restriction Technologies (GURT), better known as ‘terminator genes,’ which prevent farmers from reusing seed stocks for multiple generations [1]. According to Pew Research Center, 39% of Americans say that GM foods are worse for their health, compared to non-GM foods [2]. According to a 2016 Gallup Poll, 51% of Americans have a negative view of pharmaceutical companies [3]. ‘Big Pharma’ and ‘Big Ag’ are boogeymen that roll off the tongues of many Americans. And the stigma is even worse in other parts of the world.
A common criticism of biotech companies is that they act in ways agnostic or arrogantly unaware of what the public wants. To make sure that our project would not fall into the category of ‘well researched but completely unwanted,’ we made sure to consider from the beginning the specific cultural contexts in which our device would be used. This in turn, helped us narrow down our possible ideas, by beginning with a problem and a set of specific contexts, we were able to discard many possible project directions.
We designed a mammalian cell-based bioassay that reports activation of specific stress pathways via fluorescence, for use in environmental toxicology. To do this, we selected transcriptionally regulated target genes which are present in mammalian cells and are involved in stress pathways (see figure 1 below). We isolated the promoters with transcription factor binding sites from these target genes and coupled them to a fluorescent reporter gene. We selected EGFP, a variant of green fluorescent protein (GFP). GFP is ubiquitous in synthetic biology due to its reliability and ease of measurement [4]. EGFP is derived from GFP, and has been optimized for use in mammalian systems. When a chemical of concern is screened using our assay, it will trigger a specific stress response, and the reporter gene will be expressed, causing the assay to fluoresce. The fluorescence of the assay can be quantitatively measured and analyzed. This assay will provide data on the effect of chemicals of concern on the physiological health of mammalian cells; measurements may be easily taken a range of concentrations, durations of exposure, salinities, pH, temperatures, nutrient availabilities, and other conditions. This also allows for measurement of synergistic or interfering effects due to multiple chemicals of concern present simultaneously.
////insert table//// We selected eight promoter constructs derived from five target genes and coupled them to EGFP. These promoter and reporter gene constructs were inserted into a plasmid and transfected into two cell lines, originating in mice (AML-12) and hamsters (CHO-DG44), respectively. The resulting bioassays were exposed to five different chemicals of concern at a variety of concentrations and conditions. Although human cell lines would make a superior model for human disease, compared to cell lines derived from hamsters and mice, for our project we chose not to use human cells, for reasons of safety, reproducibility by other teams, and the constraints of the competition.
A cell-based approach cannot replace in vivo toxicology studies. However these studies require extensive funding, time, and other resources. By developing a relatively low-cost, cell-based bioassay, preliminary data may be quickly gathered, allowing for more informed decision making as to which in vivo studies are necessary. By using a cell-based preliminary assay, it is our hope that researchers will be able to quickly gather data, make more informed decisions, and save resources. Our cell-based bioassay may also be used to add to the body of knowledge concerning the effect of specific chemicals of concern on the physiological health of mammalian cells and the mechanism of stress.
We chose to use mammalian cells because they make much more accurate models for human health than bacteria or yeast. Furthermore, within the iGEM competition and the field of synthetic biology as a whole, there has been relatively little work with mammalian systems, compared to bacteria, yeast, and algae. Working with mammalian cells brings a variety of new challenges and opportunities to iGEM: they are more difficult and expensive to culture than bacteria, they require specialized equipment and safety training, they can be used to produce proteins suitable for use in human therapeutics, they can be used for more complicated circuits and pathways utilizing spatial/temporal differentiation, and they are much more sensitive to chemicals in the environment (allowing for more sensitive biosensors and bioassays).
The tribe have reported unusually elevated rates of cancer and miscarriage incidence, and have indicated they have reason to suspect that the cause may be tied to environmental pollution on their tribal land from local agricultural and forestry corporations. Researchers from UC Davis have been collaborating with the tribe’s scientists and governing council to gather data pertaining to environmental and human health.
The agricultural and forestry corporations in the region surrounding the tribe’s land are currently operating within legal regulations, however the tribe has indicated that these regulations are not as strict as they would like. One example a tribal member provided was that currently, herbicides may be applied within fifty feet of sources of drinking water. A concern is that this distance is not sufficient to prevent contamination of drinking water supplies. A variety of harmful chemicals have been found in the waters of the tribal lands, particularly microcystin toxins and organochlorine pesticides [12]. Analysis of water samples by the Young Lab at UC Davis in 2017 also found the presence of low concentrations of pharmaceuticals, including warfarin, in the waters of the tribal lands.
We were informed by tribal members that the members of the tribe interact with the flora, fauna, and water of the region regularly. These interactions take forms including the consumption of seafood, particularly salmon and shellfish, harvesting local plants and processing them to make baskets, swimming in the river, and ceremonies involving locally harvested materials.
If the working hypothesis is found to be supported, that the tribe’s health crises are linked to environmental pollution of their lands and water, then the remedy would be to tighten regulations concerning the use of pesticides, herbicides, and other potentially harmful compounds. To affect such a significant policy change would require substantial scientific evidence, including careful in vivo studies. Our bioassay can serve as a tool with which to quickly and relatively cheaply acquire data which can be used to identity areas necessitating further study and inform which specific results to expect.
If this working hypothesis is not supported by further study, alternative explanations for the tribe’s health crises should be explored, including predisposing genetic factors within the population and other factors. This possible explanation– that the tribe may experience elevated rates of specific diseases due to genetic predisposition– was suggested during the visit to a member of the tribe’s scientific body by a member of our team, and elicited a surprising response. The tribe’s scientist indicated that such a hypothesis would be very negatively received by the members of the tribe, for historical and cultural reasons. It would be perceived, the tribe’s scientist said, as an attempt to blame the tribe’s health problems on an inborn deficiency of the members, which given the persecution and genocide which the tribe underwent in the nineteenth and twentieth centuries, would strike a very powerful and negative chord. The scientist went on to reference several well publicised cases in which geneticists have squandered the trust of indigenous communities [13].
We selected several major environmental contaminants in the region for use in our project: copper sulfate, warfarin, 2,4-D, and metam sodium. These chemicals were selected based off their prevalence in the region, their negative impacts on human health, and the availability of use for our project. Additionally, we included hydrogen peroxide as a positive control, as it is easily obtained and causes oxidative stress to cells.
“In this course, which will be preferably offered on-site in Yurok Country and feature YTEP and other Yurok tribal presenters, university researchers will be trained on tribal sovereignty, tribal intellectual property rights, trial history, tribal land and water rights, and on building effective collaboration with tribes. While the focus will be on working with the Yurok Tribe, the principles taught will be applicable to collaboration with other tribes and indigenous nations in the US and globally.”
At the site visit, the other researchers and our team were taught some of the history of the tribe by a member of the tribal leadership council. We were also taught some of the customs and ceremonies which the tribal members perform, particularly regarding the strong cultural connection between the members of the tribe, the land of the region, and the native flora and fauna.
The team of UC Davis researchers included several professors of Native American Studies who helped the other researchers in questions of best practices regarding cultural competency in the course of their research. A key point of best practices we were told was to never claim the authority to say what another group does or does not believe, unless it is approved or previously expressed by an authority of the group. For example, it would not be acceptable to write on our iGEM wiki “the _______ tribe believes _______, therefore...” Any claims made about the beliefs or opinions of another group should be thoroughly grounded in verifiable evidence that the group does in fact hold these beliefs. For example, a claim may be supported by quoting a resolution or law passed by a body such as a tribal leadership council or another authorized body.
At the first meeting, our team introduced ourselves to our fellow researchers, the tribe’s chief scientist, and members of the tribe. After we explained who we were and what the iGEM competition was, the tribe’s chief scientist said that she was familiar with some work being done regarding synthetic biology and bioremediation, and that a project involving release of genetically modified organisms would not be acceptable to the tribe. The iGEM competition as a whole has strict ‘NO RELEASE’ rules, however many projects are designed in such a way that the ultimate use-case for their product would require intentional release into the environment.
Through the course of the cultural competency training and the site visit as a whole, a theme that resurfaced many times was a history of the tribe being taken advantage of by outsiders. Possibly due to this, many of the tribal members expressed skepticism of the intentions of outsiders. To paraphrase a comment made by a tribal member, “Plenty of people come to a tribe from a fancy university promising to fix things, and then they publish a few papers, get a few more letters after their name, and leave, but the problems haven’t gone away.” We were duly aware of this sentiment throughout the site visit, and took measures to ensure that we did not promise anything we could not fulfill, given the limited time and resources of the iGEM competition.
Interestingly, within the ordinance, the tribe makes several exceptions. The first is unusual: “Genetically engineered or modified organisms do not include organisms created by traditional selective breeding, [...] or microorganisms created by moving genes or gene segments between unrelated bacteria” [14]. As much of biotechnology and synthetic biology uses bacteria as a host, we were surprised to find that the ordinance deemed the majority of the work done in the iGEM competition as acceptable.
The ordinance also provides exceptions to the prohibition for “State or federally licensed medical research institutions, medical laboratories, or medical manufacturing facilities engaged in licensed medical production, or medical research involving genetically engineered or genetically modified organisms,” as well as for, “Educational or scientific institutes” [14]. This makes it appear that the major focus of the ordinance is to restrict agricultural biotechnology firms and their crops/livestock on tribal lands. The ordinance specifically refers to transgenic salmon– which are referred to as a threat to their way of life– and the significance of the wild salmon to the tribe’s cultural values.
We carefully considered the position taken by the tribe, concerning the introduction of genetically modified organisms as a threat to their cultural values and traditional way of life. While many arguments made by opponents of GMOs focus on perceived threats to human health, which can be settled empirically by careful in vivo studies, cultural arguments cannot be dismissed as easily. A community should have the right to live according their values and uphold traditional ways of life. If certain communities decide that their values are incompatible with the introduction of genetically modified organisms on to their land, then their decision should be respected.
The visit with the Native American tribe helped us focus our project and become aware of different sets of legal frameworks in which we operated. While working on the campus of our university, we were subject only to federal (America), state (California), and local (Yolo County, City of Davis, University of California) laws. If we were to return to test our device on tribal lands, we would be required to follow their specific ordinances and regulations, including seeking prior written permission to use genetically engineered devices for biomedical research. Likewise, it would be necessary to seek prior written permission before testing environmental samples taken from tribal lands. A similar procedure would be required when working with other communities.