Team:East Chapel Hill/HP/Gold Integrated

Communication with Assistant Professor Randy Stockbridge from the University of Michigan

One thing our team learned in the lab is that science isn’t always predictable and our experiments were certainly no exception. We made an unexpected observation from our experiments characterizing the growth of the ∆-crcB knockout E. coli, (basically wild-type E. coli with the fluoride channel knocked out):

• ∆-crcB can grow on high concentrations of fluoride (1-2mM) provided there is a wild-type strain growing on the plate next to it.

Our mentor helped us hypothesize some reasons why these observations could be occurring. For the first observation, we hypothesized that either the growing E. coli are secreting an essential metabolite, or, there is a fluoride scavenging mechanism in E. coli.
To seek helpful insight as to our observations, we emailed Randy Stockbridge, an Assistant Professor from the University of Michigan who has expertise in fluoride toxicity/protection mechanisms in E. coli, who told us everything she knew about fluoride toxicity. We learned that it is intimately linked to environmental pH, meaning much more fluoride enters the cell when the pH is low (HF crosses the cell membrane more readily than the fluoride ion). Dr. Stockbridge suggested we try doing serial dilutions on plates that were highly buffered to see whether there was a possibility that the phenotype is pH linked. Her prediction turned out to be spot on! Our updated results illustrated that buffering the plates results in a delayed growth of the delta-crcB strain on 1mM F when grown near WT, compared to the unbuffered plates.

Interview with Hydrogeologist and Water Quality Specialist Laura Craig

Q: On August 8, 2017 our team had a skype conference call with Laura Craig, the author of “Recommendations for fluoride limits in drinking water based on estimated daily fluoride intake in the Upper East Region, Ghana” - an important document for our team during the early stages of project development. Ms. Craig has a background in water resources and water quality. She also lived for a year in a rural community in Ghana where she observed daily life and worked on finding cheap ways to remove excess fluoride from drinking water. To see the questions and answers of the interview, continue reading below.
In your study, you mentioned that “water sources are decentralized and all residents rely on community hand-dug open wells, or borehole drilled hand-pump wells”. I understand that the borehole drilled hand pump wells are the community’s preferred source of getting drinking water because groundwater from handpump wells contains less sediment, is less prone to microbial contamination, and the water is easier to collect. However, they also have “the highest average fluoride concentrations (3.4–4.5 mg F− L−1 )” and you predict that there will be many more cases of severe dental and skeletal fluorosis in the coming years (because these systems were installed between 2008 and 2013). Why do these borehole drilled hand-pump wells have so much less contamination in terms of sediment and bacteria yet they have higher fluoride concentrations than the hand-dug open wells? Can you describe how people collect and allocate the water they need for cooking, cleaning, bathing etc. within this community?

A: The borehole drilled hand-pump wells are DEEP - which means that water and rain (microbial contamination) seep deep down into the ground - and through that journey, a natural filtration process occurs which filters out the sediment and other nasty microbes. The bad thing about these borehole drilled hand-pump wells is that they are more prone to high levels of fluoride which comes from erosion of the rock. On the other hand, the hand-dug open wells are more susceptible to microbial contamination because they are much shallower and closer to the surface. The good thing about these shallower wells is that they may not be tapped into by the fluoride rock and may even be diluted by the rain water.
Since the water system is decentralized, there is no water treatment plant like in most western countries. Most of the people in the community Ms. Craig was staying with were farmers, meaning they don’t live clustered together but instead live around their land. The lucky people in this community live close to a well and can easily get their water. Others have to walk at least 400 meters. For those who have to walk, they often will come with a cart pulled by a donkey and multiple huge jugs which they spend their mornings filling with water. This takes them a very long time; at most, even half of the day!

Q: Initially, to check the fluoride concentrations in samples of water taken from the wells, you used an Orion 4-star meter and fluoride ion selective electrode, with 1 ml TISAB III added per 10 ml sample just before analysis (APHA, 1998). What other methods of measuring fluoride / removing excess fluoride exist on the market? Are they easy to use? Are they viable for use in developing countries like Ghana?

A: Current products on the market such as the Orion 4-star meter which are designed to measure the concentration of fluoride are actually pretty easy to use as well as quite accurate - however they can be very expensive! The electrode itself is several hundred dollars and needs to be replaced each year, as well as requires some level of technical training to operate. Realistically, any filters/measuring devices currently on the market could never be implemented unless an organization paid for it. Finding a cheaper and easier way to identify the range of fluoride in drinking water is still a concern for many.

Q: The World Health Organization recommends a daily fluoride limit of 1.5 mg F− L−1, however your study recommends a dose of 0.6 mg F− per kg body weight per day as optimum for preventing severe dental fluorosis and maximizing dental caries protection by combining 0.05 mg F− per kg body weight per day from water (Institute of Medicine, 1997), and 0.01 mg F− per kg body weight per day from food (McClure, 1943). This is significantly lower than the recommended limit posed by WHO. Does the 0.6 mg/L just apply to younger children, or, in a perfect world, would you like to see this implemented for all ages?

A: It would be ideal if 0.6 mg F - per kg body weight per day was the limit for adults and children. However, the lower the limit the harder it is to reach that limit because it increases the cost of good filtration. That’s why I thought that having some sort of a compromise in which we focused this limit on vulnerable younger children (because developing teeth/bones are more prone to damage) would be more cost-effective. The flaw with this idea is that it may be difficult to have two standards - one for adults and one for children.

Q: In your study you do not address other health impacts of consuming excess fluoride, including reproductive and developmental changes, neurological effects, and impacts on the immune, gastrointestinal, renal, hepatic and endocrine systems, and carcinogenicity (NRC, 2006; Ozsvath, 2009 and references therein) because data are limited and adverse health effects are still not clear. I believe you sudy was published in 2015. Have you learned any new information regarding these health impacts? What is your opinion on controversial studies which show links between depreciating IQ scores amongst young children with consistent exposure to toxic levels of fluoride?

A: I think these potential health impacts are very important to address. Unfortunately, I cannot comment because of a lack of information.

Q: You claim that “there is no effective way to prevent fluorosis that is also cost-free”. Our aim is to turn our project into a bioreactor that can bioremediate fluoride as well as a biosensor that can detect excess levels of fluoride - all at a low cost. What do you think is the best way to make use of this mechanism, if successful? How do you suggest we can safely incorporate this in underdeveloped countries where over fluoridation is an issue?

A: A biological absorption system with an additional step for filtration seems like the best way to make use of the of the fluoride riboswitch. When I went to Ghana, water filtration companies were selling treated water in sealed sachets that people were buying for a couple cents. They used a reverse osmosis filter; and tests showed that the water they were selling had virtually no fluoride in it. You could adopt a similar model where you treat water at a plant and people come and fill up their jugs all at a low cost. That way everything is centralized and easier to control. Also, these water companies may be interested in using the cheaper method (fluoride riboswitch) rather than a reverse osmosis filter. The cheaper the production costs, the cheaper they can sell the water to the people!

Q: In conclusion, you state, “Steps forward in addressing the problem of fluorosis will need to include ongoing educational programs, beginning with the schools and health clinics, to help the residents understand fluorosis; for currently only 24% of the population in the study area understands that high fluoride drinking water is the cause of discolored and damaged teeth (data from this study)”. What sort of an educational program do you have in mind? - because this is something our team would be eager to work on creating. In other words, what is the best way to have everyone in the community understand the cause of fluorosis and have an incentive to address it?

A: When I first arrived, I found that most people did not know their water was causing fluorosis. They said the water looks great and tastes great so there is nothing to be worried about. If they don’t understand what fluorosis is within their own communities, you can’t sell the idea of TOXI-SWITCH to them. You have to start with education from within the community. You also must illustrate your idea in a way that is understandable and acceptable. For example, in east Africa they found that bone char was effective in removing fluoride. They would take cow bones and cook them so that they would char and become a powder. This would act as an adsorbent to remove the fluoride. This idea gave some people the creeps and thus was not very popular. It also gave the water a funny taste.
In these large rural communities a very oral culture is present, meaning they learn from oral storytelling and help each other through town meetings. Because government workers are not really related to the community at all - they are just assigned posts which are subject to change every few years or so, it would be best to find someone from within the community that likes our idea and believes it's worth trying. This well-respected community member could hold community clinics and talk to people about the causes of fluorosis.
It is important to remember that fluorosis is not exactly these people’s top priority, they are more worried about getting regular income, food, and water (they are subsistence farmers). In fact, some of them don’t even know what fluorosis is. In northern Ghana, there are virtually no cases of skeletal fluorosis - a much more obvious, severe, and crippling problem which has been documented in China and India. Dental fluorosis are the only documented cases, and dental fluorosis does not prohibit one from working, making it a mild problem.

Q: Why do some locations have higher concentrations of fluoride than others?

A: Fluoride comes from the rock. In Ghana, there are pockets in the earth rich in “bongo granite”, which contains fluorine. As the water travels through the ground to a distant area, fluorine starts to disperse and dilute itself. In other countries where the concentration of fluoride is higher than in Northern Ghana, the high fluoride rock is more widely dispersed.
That’s why one of my other ideas was to install wells a little farther away from the water pipe, which would be in a high fluoride concentration area. However, the initial cost would be very high, somewhere between $5,000-$10,000, because of the pipe system and having to pump the water mechanically. But once set up, it can maintain itself for many years.

Q: What are other ways people can be exposed to fluoride?

A: People can be exposed to fluoride by breathing in air pollution from industry, drinking tea (tea leaves are very high in fluoride), and eating certain foods - whose concentration depends on how much fluoride is in the soil and certain types of pesticides.

Ethics in Synthetic Biology Reddit Forum

Along with the iGEM teams of Duke and Gaston Day school, we created a subreddit to initiate discussion about synthetic biology. Our team, the Duke, and Gaston Day teams function as the moderators of the Reddit thread called r/iGEM_Exchange. The thread was allowed us to engage with the public in discussions about ethical implications of synthetic biology. Contributors had an opportunity to talk with others about pressing current issues like GMOs and CRISPR. With this thread, we are able to reach out to the public and share our knowledge about genetic engineering and iGEM and their social impacts (and of course, to share the iGEM memes).

Tasty-Style Videos

Taking inspiration from Buzzfeed’s Tasty videos, our team created a playlist of short lab technique videos with contribution of other iGEM teams: iGEM Peshawar 2017, UNBC iGEM, and TNCR Korea. While we created a video on serial dilutions, they created videos documenting the lab techniques of transformations, agarose gels, and ligation prep. Our intention in creating these videos was to teach viewers about common lab techniques in a simple and engaging way to aid them in their scientific endeavors! We wanted to give assistance to the group of scientists who may be new at lab techniques, and give the public an idea of what happens in the lab.