Human Practices

Project finding

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The lakes in the town of Tuebingen and its surroundings sometimes grasp the attention of people in the summertime due to their green color. This is the case for many bodies of water elsewhere too and the reason for this discoloration are usually algae or cyanobacteria. Sometimes you can also discover warning signs at the shore of those lakes warning people not to swim in them because of the toxins the cyanobacteria produce when they occur in high numbers, in so called algae blooms. These cyanotoxins could not only potentially harm humans that swim in the lakes but also animals who might drink the contaminated water. Due to the possibility of algae bloom occurrences increasing in the future because of accumulating nutrients in water and rising temperatures1, we, the iGEM team Tuebingen, decided to make the detection of a particularly dangerous toxin, Anatoxin-a, easier.

Although it would be great to build a biosensor for many different cyanotoxins, we chose Anatoxin-a, since it is not well researched yet. We therefore hope our biosensor could help in gathering more information on Anatoxin-a and its effects. We became interested in this topic due to the effects it has on the environment and the danger cyanobacterial blooms can pose to ecosystems.

From the beginning when our team was first introduced to the topic of Human Practices we soon realized the questions we would face: How exactly our project would be useful for the world and whether it would be safe to implement. First we discussed who the parties involved in our project would be. It broadly would be people with pets, especially dogs, farmers, since their cattle might drink water from outside sources as well and people swimming in lakes. More specifically we thought of testing facilities for cyanobacterial toxins, as they would be the ones to use our biosensor in the end. Hence we wanted to contact experts on the topic and also the public to receive feedback and to share information about our project.

We also wanted to learn about how knowledgeable people were about this topic in general and how they felt about using genetically engineered organisms in biosensors, since this can be an issue a lot of people are hesitant about. We also thought about how people would feel about developing a biosensor for such a dangerous toxin. Based on the replies we wanted to think about further measures we would be able to take.

Planning phase

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But first of all we decided to ask several experts for help concerning the exact details and safety management of our project. Firstly, we talked to Dr. Karl Forchhammer, who is a microbiologist particularly researching processes in cyanobacteria. He was convinced of our project idea but gave us to consider that the quantities in which Anatoxin-a occur and are deadly are little and therefore our biosensor had already to work with small amounts. He also offered us the lab space we used to conduct our experiments because he and his teams are researching cyanobacteria as well. Through him and his lab we also learnt more about safety measures we had to pay attention to when working with genetically modified organisms in general and a dangerous toxin like Anatoxin-a specifically. Dr. Iris Maldener was the one giving us a safety introduction and who we could go to for questions about specific measures. Sadly, we could not test our biosensor with Anatoxin-a in the end, due to issues with the shipment, like mentioned in the project description.

We then got the opportunity to talk to Dr. Joachim Schultz about our project idea. He is a pharmaceutical chemist who most recently worked on signal transduction through cAMP in bacteria and researched properties of receptors of membrane domains of human adenylate cyclase. He gave us advice with the experimental design of our project concerning the chimeric protein using chemoreceptors and the two-component system by recommending us known authors, reviews, and papers. We also talked about HAMP characterization (a domain that is present in Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases2) and he even gave us tips for presenting scientific topics, which proved useful when talking to sponsors and when we would have to present our project in general.

Dr. Victor Sourjik is a microbiologist whose team’s focus is on quantitative analysis of the organization and molecular and physiological functions of cellular networks in microorganisms. We used publications of his group to plan our experimental design and also had the chance to contact him personally. He offered us advice and materials we needed for our experiments: the PctD ligand binding domain for the ITC measurement, a PctD template for the cloning of our bacteria, a reporter plasmid conveying the ability to produce GFP in response to EnvZ activation, and the VS996 strain with deleted EnvZ and deleted chemoreceptors to be used as background strain. He also gave us information about the structure and affinity of the chimeric biosensor. Through him we were also introduced to Wenhao Xu who is part of the Victor Sourjik research group. She was the person recommending creating a biosensor using EnvZ and that the reporter should be a fluorescent protein.

Interview with an expert on cyanobacteria and their toxins

In one of our first Human Practices meetings we discussed and thought about the exact areas of concern for our project and tried to find groups of different people who could help us with their expertise. One person we came across was an employee of the German Environment Agency who specialized in cyanobacteria and cyanotoxins. They agreed to hold an interview with us but asked us to keep their name anonymous.

Their tasks in the agency mostly relate to drinking water hygiene, more specifically in resource protection of surface water. They are researching mechanisms in the bacteria themselves but also the behavior of their toxins in different water conditions. They also look into the regulation of them. Especially in recent years they worked with anatoxins. We also asked them about their water testing procedures. They use different methods of chemical analysis and examine the toxins qualitatively and quantitatively.

Next we asked them about the conditions under which cyanotoxins are produced. They told us that cyanobacterial blooms which is the time when the toxins occur in higher abundance are linked to an increase in nutrients in the body of water, especially phosphorus. A rise in temperature can also play a role. Furthermore we asked about the danger cyanotoxins pose and they replied that microcystins, another type of cyanotoxins, are especially dangerous when one is exposed to them chronically. Neurotoxins like Anatoxin-a are dangerous when acute intoxication takes place. But all of them are only dangerous when swallowed directly. This could also become a problem for people swimming in lakes when swallowing water accidentally, but especially for smaller children.

Anatoxin-a itself is especially dangerous to dogs ingesting water contaminated with it, although the exact reason is unknown. Also, there are no thresholds for Anatoxin-a in waters in most countries, although it is possible that the toxins might become a bigger problem in the future. In Germany, Anatoxin-a is not a big problem yet but there are other countries, like New Zealand for example, where the issue is more present. Lastly, they suggested we look into dihydroanatoxin-a as well, which is a lot more toxic than Anatoxin-a but has the same mechanism of action.

We gained a lot more information about cyanobacteria and their toxins in general through this interview. It raised the issue of algae blooms possibly increasing in the future, if too many nutrients accumulate in bodies of water, as well as possibly due to rising temperatures because of climate change. It is not sure whether this is the case for all bodies of water, which is also a reason to monitor the existence of cyanobacteria and Anatoxin-a is important.

We also discovered that Anatoxin-a and its exact effects are not well researched yet which we hoped our biosensor could help with. Also, by learning about their job as a person surveying water quality and checking for toxins, we became interested in visiting testing facilities ourselves.

European Meet-Up in Hamburg

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In the beginning of July we were invited to join the iGEM European Meet-Up organized by the Hamburg team this year. By attending the event we got the chance to get to know other teams and their projects by having poster sessions. Every team had to prepare a poster in advance that got exhibited and people could go to to ask questions. Furthermore we could talk to them about ideas for sponsoring and had the chance to plan collaboration. We also had the opportunity to take part in workshops, for example the Human Practices workshop, in which everyone collected ideas on who stakeholders or concerned parties for their project were and how to collaborate with them.

Overall the meeting made us get new ideas for our own project and we hope we could help out other teams as well. Thank you again to team Hamburg for organizing such an exciting meet-up!

Visits to the ministry for public health and a chemical testing facility

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After talking to an expert on cyanobacteria and toxins we got the idea of visiting places testing water samples for them. We decided to visit health authorities and testing sides in our state to present our project because they would be the ones making use of our biosensor in the end. On the 24th of August 2022 we visited the central public health authority of Baden-Württemberg (Landesgesundheitsamt Baden-Württemberg). Dr. Fleischer, head of the department for water hygiene, showed us around and explained the techniques they use to analyze water.

First, this meeting brought us further in understanding the procedures the ministry uses to take samples, analyze them, and generate the report. We learned that the way the sample taking is done can strongly impact the results since bacteria are not homogeneously distributed in the water. And since sample taking and laboratory analysis are not done by the same people, in some cases it might lead to false results.

Moreover, we learned about the water map of Baden-Württemberg, where anyone can check the state of the lake or river they want to swim in. It doesn’t work perfectly because of some software and data policy issues. Talking about lakes in Baden-Württemberg, we also discovered lakes contaminated with cyanobacteria about which we didn’t know.

Regarding the measuring methods, we found out about measuring the chlorophyll A levels instead of measuring the produced toxin itself. Prominent levels of chlorophyll A indicate possible cyanobacterial contamination.

At the end of the meeting, Dr. Fleischer also suggested us visit the chemical facility of the ministry in Sigmaringen to get a better understanding of the sample taking and evaluation processes.

Regarding our biosensor, he said that he thinks it might be useful if the sensitivity is high enough. Optimally, one would have a biosensor which is able to detect not only Anatoxin a, but also other cyanotoxins like microcystins. Due the autofluorescence of chlorophyll, he advices us to use RFP instead of GFP as a reporter.

On the 29th of September we visited the chemical testing facility or CVUA (Chemisches und Veterinäruntersuchungsamt) in Sigmaringen like Dr. Fleischer suggested. The CVUA is mostly responsible for the analysis of drinking water for toxins occurring most often.

They told us about their current way of testing water for Anatoxin-a and other toxins which might be contaminating the water. They use the HPLC/MS method where it takes around 24 hours for one measurement of up to fifty samples. Each sample is tested for multiple toxins including Aflatoxin A and hydroanatoxin-a. The calibrations preceding the measurement take the longest and it is also needed to lyse the cells through multiple freeze cycles. A typical week of testing water samples might look like this: If the samples are taken on Monday, they will be evaluated by microscopy for the presence of cyanobacteria by the Health Ministry until Wednesday. The analysis can then start on Thursday and will be finished on Friday, which is also the day the report must be finished.

Therefore if we released our biosensor as a kit it would be useful in the first stage of testing when deciding what samples should be analyzed more closely and to shorten the waiting period.

We also asked about the possibility of misusing our biosensor with wrong intentions. They told us the possibility our project could be abused is low, since the amount of Anatoxin-a in water which is needed to hurt a human is relatively high. For animals like dogs however, this could be different.

Public survey

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We then decided to further explore the possibilities of our biosensor being misused and decided to conduct a survey in the city of Tuebingen where we would ask people on the street for their opinions on genetically modified organisms and synthetic biology in general and more specifically whether they’d feel safe if a biosensor like ours would be used and if they’d think it’d be helpful.

There are no concrete guidelines by our university concerning public surveys so we made sure it complied with the ones of the city of Tuebingen. We asked for the people’s participation and told them what the survey was about beforehand. We then also asked for their permission to publish their answers anonymously.

First we gave an introduction to who we were, what iGEM was and why we were interested to hear their answers.

The exact questions we asked looked like this:

The results were as following: many people at least sometimes enjoy swimming in lakes and a majority of them also concern themselves with water contamination; some of them specified they were looking out for drugs or medicine and pathogens in bodies of water.

After we gave them an explanation of the topic of synthetic biology, we asked them to give us associations with the topic. Most of them weren’t familiar with the topic but they had associations like ‘environment,’ ‘genetic engineering,’ ‘bionics,’ ‘genetically modified food’ and ‘microbiology.’ On the topic of whether people had concerns about the application of synthetic biology the answers were split: Many of them did not have any worries with its application but some were concerned, more specifically with genetically modified food and the impact it has on the agriculture.

Then we asked the questions concerning our biosensor. Most of them could imagine a way in which the Anatoxin-a biosensor could be used, especially when testing drinking water. All of the answers were positive that our project would be useful. Most of them also agreed, that there are no dangers in the usage of such a biosensor, but some expressed their concerns that the modified bacteria could escape and pose a threat to ecosystems or transfer mutations to other organisms. A majority of people replied that they might inform themselves about the issue of cyanotoxins more but only half of the people we asked were interested in attending an event surrounding synthetic biology.

We learned a lot through this survey about our own project and people’s view on synthetic biology in general. We gathered that most people were not well informed about the dangers cyanobacteria and their toxins could pose and were therefore hoping that participating in the survey awakened interest in them. Or there could be more information we give out concerning topics around cyanobacteria, like their algae blooms and the threat they can pose in ecosystems when our biosensor will be applicable. It also made us more aware of the fact that some people would be concerned with the usage of a biosensor utilizing genetically modified organisms. Since our biosensor would be used in a lab and only the water probes would be gathered in nature, this would not cause issues, however, it reminded us of the rigorous safety management rules around dealing with genetically modified organisms. But in general we think that more information about GMOs offered to the public would prove useful, especially how they are overseen in the lab and that they are not released in the environment uncontrolled. The improvement we thought of could be that we could go to lakes specifically to ask people there and also pay more attention on asking different age groups. Also we could design the survey to be more interactive so people have something they can immediately take away from their participation.

Planned talk with Dr. Thomas Potthast

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Because we as a team wondered how our project might be abused in the sense of dual use, since we worked with a dangerous toxin, we wanted to talk to an expert about this topic. Therefore we asked Dr. Thomas Potthast of the university of Tuebingen to hold an interview with us. He is an ethics professor who specializes in biological sciences. The interview will take place on the 15th of October and we will publish the answers to our questions via an info post on Instagram. The main questions we want to know more about are the definition and ambiguity of the term ‘dual use,’ in what way our project or the distinct stages of our project could harm people or be used to harm people and what options there are for us to prevent something like this. We are looking forward to meeting and speaking with him soon.

Concluding thoughts

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Overall, everyone we talked with and all the experience we got helped us to think more exactly what our project is about and specify who the people concerned by this topic are. We got helpful input on how the implementation of our project could look like and what more needs to be done for our project to be finalized. It also made us more aware of problems that might occur in the future when further developing our project and concerns surrounding it and gave us ideas on how to solve them. We learnt what is necessary to make bodies of water safer for everyone enjoying them and hope that the biosensor we developed will contribute to that.

Thank you again for everyone willing to meet with us and helping us with our project!