Team:Brno Czech Republic/Human Practices

Human practices

Introduction

Due to the changes in the environment associated with human intervention, cyanobacterial overpopulation is affecting people and ecosystems all over the world. In the project we are trying to find a solution for disposing of the problem with cyanobacteria overpopulation at Czech Republic. For that reason our Human practises focused mainly on communicating with people about their experiences with cyanobacteria. As for the communication with the experts, firstly we had to find out the situation with cyanobacteria in the Czech Republic and the problems associated with them, among other things we also chose an organism with which no one at our university had experience and therefore we had to find an expert at another workplace.

Communication with experts

We've extensively discussed the technicalities of our project with our professors and experts from our university, for which we are extremely grateful. Special thanks belongs to Bacillus subtilis expert Dr. Libor Krásný from the Czech Academy of Sciences and cyanobacteria expert Mgr. Geriš Rodan from the Povodí Moravy, s.p.

Consulting our project with experts from Masaryk University

First we would like to mention our PI - Mgr. Pavel Dvořák, PhD., Mgr. Karel Říha, Ph.D. and Ing. RNDr. Martin Marek, PhD., with whom we discussed the design of our project and the course of work during wet lab experiments, where we are grateful for all the advice and recommended modifications to the project that they gave us.

We also consulted our project with other experts from our university, who were interested in our project. Each of them provided us with valuable advice and ideas that we could implement to our project in the future.

Prof. RNDr. Jiří Doškař, CSc.
Prof. Jiří Doškař is the head of the Section of Genetics and Molecular Biology of Masaryk University, as well as the leader of the Laboratory of Molecular Diagnostics of Microorganisms at the same institution. His work is focused mainly on horizontal gene transfer, bacteriophages, their interaction with Staphylococcus aureus and the phage therapy. He teaches some of the most important courses in the Department of Experimental Biology at Masaryk University.

Prof. Doškař thinks we could use cyanophages or their proteins to lyse cyanobacteria. He fears that usually used lysozymes won’t be effective against the atypical cell wall of cyanobacteria. According to his opinion, the cyanophages would not have to be GMOs at all, but if needed, we could engineer them to produce more of the target proteins. We had thought about following his advice and discussed it with our PIs but in the end we decided against it because none of us had any experience with work on phages. We were also encouraged by the work of iGEM team Peking 2014 which proved that the lysozyme from Gallus gallus is able to lyse the cyanobacterial cell wall. However, we keep his idea in mind and if the way we chose proves to be a dead end, we will come back to the cyanophage strategy that he proposed.

Prof. RNDr. Zbyněk Prokop, Ph.D. and Mgr. David Kovář, Ph.D.
Prof. Zbyněk Prokop is the head of Kinetics and Microfluidics laboratory belonging to the Loschmidt Laboratories of Protein Engineering at Masaryk University. His projects are focused for example on enzyme-based technologies, biosensors and machine learning in enzyme engineering. Dr. Kovář works at the laboratory led by prof. Prokop and focuses mainly on microfluidics and biosensors. We talked about the project with both of them together and here we summarize their opinions because they agreed with each other practically about everything.

They both think it is great that our proposition solves many problems, not just one. They recommend buying the hardware parts of our device from a company that builds home water-cleaning devices or having it built by someone at BUT. Following this advice, we addressed two students from BUT (brothers Petr and Pavel) and they joined us as leaders of our IT team and they also took care of the design of our device.

Consulting cyanobacteria situations in Czech Republic with Mgr. Geriš Rodan:
To get more insight about cyanobacterial overpopulation and its adverse effect in Czech Republic, we contacted Povodí Moravy, s.p., which manages and maintains watercourses and water management facilities in the Morava river basin. There we've spoken with Mgr. Geriš Rodan.

Mr. Rodan is an expert on water analysis with a focus on algae and cyanobacteria. We first compared the situation around cyanobacteria in other European countries. We found out that one of the factors contributing to cyanobacterial overpopulation, other than the large amount of phosphorus released into nature, is also geographical location. For example, in Austria and Slovakia, cyanobacteria are overpopulated to a much lesser extent as these countries are more mountainous than Czech Republic. Then we discussed what measures are being employed by other countries. Unfortunately some of the methods are too expensive to be used in Czech Republic.

We also talked about the negative effects of cyanobacterial overpopulation on nature and the economy. The overabundance of cyanobacteria is not only problematic because of cyanotoxins, but also the odor associated with the decomposition of biomass, which drives people away from the affected waters. Reduced water visibility also negatively impacts diving birds as they are therefore unable to hunt fish.

Lastly, we discussed the use of our device. Mr. Rodan advised us to use our device mainly in natural water bodies as they are smaller in size. He also gave us contact on some people with whom we could consult our plans to also accumulate phosphorus.

Consulting our project with Bacillus subtilis expert Dr. Libor Krásný:
We decided to use Bacillus subtilis for our project as it fitted all of our criteria. However there was no one at our university with previous experience working with Bacillus subtilis. Luckily, we were able to contact Dr. Krásný, who has been working with this bacterium for many years and was willing to provide us with guidance and share his experience.

He oversees the Laboratory of Microbial Genetics and Gene Expression at Czech Academy of Sciences. He helped us pick and receive an optimal B. subtilis strain and plasmids suitable for our project. He also shared his experience working with B. subtilis and provided us with some of the protocols they routinely use. He advised not to store B. subtilis in the fridge and gave us many tips in regards to its transformation. We were also able to contact his laboratory when working in the wet lab to discuss our difficulties. For example, we were by mistake selecting Bacillus transformants with ampicillin, but after consultation we found out that ampicillin is not working on Bacillus and that we must switch to chloramphenicol or erythromycin.

Thanks to Dr. Krásný and his colleagues, we were able to adjust our lab work and the whole project for this amazing bacterial strain.

Communication with people

One of the main goals of the Human Practises aspect of our project was to determine whether our project deals with problems affecting the general population and what is the stance of the general public on this issue. To examine public opinion and their experiences with waterbloom, respondents were asked about these three primary areas:

Have they ever suffered inconvenience due to algae waterbloom?
How do they feel about our use of GMOs?
What aspects of our prototype should we prioritize?

A total of 11 questions were answered by 544 respondents, most of them were close to us in age (18 - 25 years old). We found that the vast majority of respondents encountered waterbloom and had to restrict their recreational activities because of it. Most respondents also had a positive opinion regarding our use of GMOs.

The answers show that waterbloom has negative effects on people’s lives, and therefore our project targets relevant issues. When creating this questionnaire, we complied with all legal and ethical requirements as well as the recommendations of iGEM (see ch. Legal and ethical aspects).

Methodology

The questionnaire was created with google forms and promoted on our Facebook page, related Facebook pages, and by our Facebook friends. We left it open for 14 days from 2.9. to 15.9. 2020.

The questionnaire was divided into five sections:

In the first section, we made sure that the participants gave informed consent before continuing.
The second section served as a differentiator between those who had encountered waterbloom and those who had not. Those who had were delegated to section three to further investigate their encounter; the remainder was delegated to section four.
The third section asked those with previous experiences with waterbloom whether and how often waterbloom restricts their recreational activities.
The fourth section asked respondents about their feelings around GMO use and about the design of our prototype.
The fifth section asked about the respondent’s gender, age, and contained a feedback box.

Results and discussion

Section 1: Informed consent

The first question was designated as informed consent. All (544 in total) respondents had to consent before continuing with the questionnaire. Further description of our informed consent design, as well as legal and ethical questions, are discussed in ch. Legal and ethical aspects.

Graph 1: Informed consent

Informed consent text: “I am over 15 years old, I agree with the anonymous processing of my answers for the above purposes, I confirm that I have had the opportunity to ask any question about the project or questionnaire survey and I acknowledge that my participation is voluntary and that I may suspend the questionnaire at any time”.

Section 2: Differentiation of respondents based on experience with a waterbloom

Before asking the respondents in detail about their experience with the waterbloom, we first made sure they had already encountered it. To provide clarity on what counts as waterbloom, we also attached its definition. Those respondents who were not sure or did not encounter waterbloom were delegated to section 4 (26.4%, 144). The remainder (73.5%, 400) continued to section 3 to further examine their waterbloom encounter.

Provided description of cyanobacterial waterbloom:

"Waterbloom is algae and especially cyanobacteria overgrowth accumulated at the surface, visible by eye. The algae layer resembles uniform stinky green porridge, or it can be seen as small flakes or needles several millimeters long. Cyanobacterial blooms emerge in late May and persist in water until October, usually with a maximum development in August or the first half of September. "

Source: sinice.cz

Graph 2: Respondent’s experiences with waterbloom

Section 3: Further examination of waterbloom encounter

Those who confirmed their waterbloom encounter (73.5%, 400 out of 544 total respondents) were selected for further examination of their experience.

Graph 3: Cyanobacterial pollution as a decisive factor (answered only by those who confirmed waterbloom encounter)

Graph 4: Waterbloom limiting recreational activities (answered only those who confirmed waterbloom encounter)

Graph 5: Swimming in contaminated water (answered only those who confirmed waterbloom encounter)

Discussion: Further examination of waterbloom encounter

According to graph 3, a waterbloom occurrence plays a role in choosing their recreational site. According to Graph 4, waterbloom limits the activities of 68% of our respondents at least once a year. Nevertheless, according to Graph 5, 34.5% bathed in the affected water. The results show that waterbloom restricts people and even threatens their health, as some admitted to swimming in the waterbloom.

Section 4: Use of GMO and design of our prototype

All respondents were shown this section. We first asked about their feelings about our use of GMOs, which we described in the text below:

“We designed a device that could clean water areas from cyanobacteria and phosphorus. Our technology uses a genetically modified organism (GMO) - Bacillus subtilis. We plan to equip our device with a system that will prevent GMOs from escaping into nature.”

Graph 6: Attitude towards GMOs use

We also asked if their opinion could be changed after a more detailed explanation of the use of GMOs.

Graph 7: Willingness to change opinion about GMO use

Following questions focused on the appearance and features of the device.

Graph 8: Design of the device

Answer number one (19.7%): The device should have a modern look, which does not necessarily imitate its surroundings.

Answer number two (80.3%): The appearance of the device should primarily mimic the surrounding environment.

Graph 9: Preference of environmental friendliness versus high performance

Answer number one (87.1%): I would prefer environmental friendliness.

Answer number two (12.9%):I would prefer higher performance.

Discussion: GMO use

To interpret the number scale questions, we calculated median and mode values. The median value for both graphs 6 and 7 is the same - 6. The mode value is 6 for graph 6, for graph 7 is mode value 7.

The median and mode values in Graph 6 shows that the majority of our population sample holds a positive attitude towards GMO use. However, similar values shows also Graph 7, which investigates their willingness to change their opinion on GMO use after further explanation of how the leakage prevention could work.

It seems illogical that those who claimed a positive attitude towards the use of GMOs would change their opinion to negative based on further explanation of leakage prevention. Furthermore, the first comment form a respondent in chapter 3.5 mentions this issue. Therefore, while we believe that Graph 6 represents our respondent's views accurately, Graph 7 might be less representative as the question was not clearly formulated. To improve our questionnaire, it might have been better to only ask people with negative views on GMO about the willingness to change their opinion.

Discussion: Device design

The results in Graph 8 and 9 show a strong inclination towards environmental friendliness and a natural look of our device. These preferences could be incorporated as a solar-panel-based power supply solution and the device’s color would blend into the environment.

Section 5: Age and gender, feedback

The majority of our respondents were women (60 vs. 40%) and most of them (80%) were our age - 22 to 25 years.

Graph 10: Gender of respondents

Graph 11: Age of respondents

At the end of the questionnaire, we allowed the respondents to leave us a message of any kind and to express themselves in written form. We received 75 messages, the vast majority of which were supportive of our activities. Here are the comments that directly related to the questionnaire or to our project:

The question "Could detailed explanation, how the leak of GMOs into the environment can be prevented, change your mind? Rate on a scale of 1 to 7 (1 - probably no, 7 - probably yes)" seems illogical. It only makes sense to me if the answer to the previous question is no.

It is not described what the device would look like - how big it would be, how would it work. It also depends on whether it is placed in a swimming pool or in an ordinary pond. It is not necessary to reduce cyanobacterial population completely; another case is recreational water areas. If the device was placed in nature, I am in favor of a landscape-adaptable appearance. If it were on a recreational site, I wouldn't mind the modern look at all.

I would like to know how you want to prevent the release of GMOs into nature and avoid pollution ... and what is its success rate...

Section 4 question 2: The explanation of the function would not change my opinion, but I would welcome the description (as detailed as possible).

Its appearance should blend in with the environment in order to not attract excessive attention from people who disagree with the idea.

The effect of cyanobacteria is not entirely negative. Yes, in bodies of water where people are bathing, they are not acceptable. But still, they convert CO2 into oxygen. Some species are even used to remove phosphorus from the water. Some have many useful properties and produce quite interesting substances (Omega3 and six fatty acids, medicinal substances ...). I would think about where it is appropriate to use this device and if it's somehow possible to process the captured cyanobacteria. Of course, I don't know how your device works, but it could be interesting.

Discussion: Age and gender

As shown in section 5, graphs 10 and 11, our respondent’s sample is not representative. Most of our respondents (75,2%) were aged 18 - 25 years. The reason for this could be that the questionnaire was mainly promoted via Facebook amongst our friends and on university Facebook pages. Therefore, it is important to keep in mind the results are mostly representing the answers of people our age. For future questionnaire efforts, we should promote the questionnaire by other means, f.e. through local communities and companies to reach a more representative sample of our society.

Discussion: Feedback

The respondents' feedback showed the necessity of properly communicating the device's appearance and its mechanisms.

Feedback 1 points to the ambiguity of some questions in our survey - elaborated above in ch. GMO use.

Feedback 2,4,5 discusses the appearance of our device. When being used in a swimming pool, the device can have a modern look. However, when placed in nature, it should be unobtrusive. Therefore, we could propose diverse designs for different uses.

One respondent suggested we should make our device unobtrusive as certain people disagree with our idea. While we do not wish to deceive anyone and we will definitely be upfront about using it, this comment helped us realize that designing a device which is not immediately apparent might help prevent acts of vandalism or theft.

Feedback 3 wants to know more about the GMO leakage prevention mechanisms. We need to be able to describe the leakage prevention mechanism in detail.

Feedback 6 points out the positive aspects of the presence of cyanobacteria and asks whether we could harness their nutrients. This helped us realize that we should definitely communicate better the goal of our project - it will never be able to completely eliminate cyanobacteria from a sizable body of water and it isn't supposed to do that. Our aim is the reduction of its population size. The accumulation of nutrients from lyzed cyanobacterial cells is an interesting idea and it is something we might look into in the future.

Legal and ethical aspects

We made sure that our questionnaire complied with legal regulations and ethical standards. The university's ethics committee’s approval is required only if the project uses biomedical methods (magnetic resonance, blood collection, etc.). The ethics committee’s approval is recommended if children are the subject of the study or if sensitive personal data is collected (health information, sexual orientation, etc.).

Our project does not use biomedical approaches, and the questionnaire itself was not aimed at children. We also do not ask for sensitive information. In our case, a consultation with the Rectorate’s Legal Department regarding consent to participate in the questionnaire and consent to the processing of personal data was sufficient. The consultation showed that we do not fall under the European GDPR on personal data protection due to anonymous data collection, as we are not able to trace specific respondents. We have also limited the respondents’ age to at least 15 years old, at which partial legal responsibility is acquired in our legal system, and we can thus consider them eligible to answer the questions in our questionnaire.

According to the recommendations and requirements of iGEM and general ethical standards, we emphasized that the questionnaire is purely voluntary, that it can be stopped at any time, and what will be the use of obtained data. The respondents had to confirm that they've read and understood the text below in order to complete the questionnaire, see section 1.

Dear respondents,

On behalf of the Generation Mendel student team, we thank you for your participation in this questionnaire. Our team took part in the international iGEM competition, in which students use the tools of synthetic biology to solve a wide range of local and global environmental problems affecting today's society.

We designed a floating device that can clean water bodies from cyanobacteria and phosphorus. Your answers will help us better understand the public's views on this issue, which we will be able to further incorporate into our project.

The data obtained from your answers are entirely anonymous (i.e., your identity cannot be traced back) and will be processed/published exclusively for the purposes of the iGEM competition. Completing the questionnaire is voluntary, and you can quit at any time.

If you have any questions, do not hesitate to contact us at igembrno@gmail.com

More information about our team and project: generationmendel.cz

You need to be 15 years old to participate in this survey.

Summary

We conducted an online questionnaire with 544 respondents, most of them our age. The results show that the waterbloom presents a problem for our respondents, as it restricts their recreational activities. We also found that our GMO solution is not viewed negatively. We were given valuable feedback, showing that people are interested in the details of our project, and it is necessary to present our project carefully. In the future, we can try to address a broader sample of respondents to make sure we represent the attitudes of Czech people more accurately.

Integrated human practices

We did the first step into completing this medal criterion right at the beginning of our iGEM journey. Our team came up with four different ideas on what our project could be about. Those ideas were concerned with getting rid of the European spruce bark beetle, creating synthetic eggs using bacteria or yeast, cleaning surfaces from brominated flame retardants and finally, cleaning water from cyanobacteria and their toxins. We researched those ideas and presented them to seven leading researchers at our university (Dr. Říha, Dr. Marek, Dr. Dvořák, Dr. Botka, Dr. Kovář, Prof. Prokop and Prof. Doškař). They gave us valuable advice and helped us to decide on the topic of our project. Some of them even decided to become our PIs and join us on our iGEM journey!

When the topic was decided, we started to design the specifics of our project. We also managed to meet with experts on cyanobacteria (Dr. Rodan) and Bacillus subtilis (Dr. Krásný) and present our design to them. After talking with the experts, we implemented some of their ideas to CYANOTRAP. We changed the design of our synthetic sequences based on what Dr. Krásný told us. When he told us he can provide us with integration plasmids for two chromosomal loci, we modified the sequences so that they can be connected into two operons. We used different RBSs in front of genes that will be expressed from one promotor to optimize their translation rate. We also take into account the advice from Mr. Rodan, who told us that the best place to implement CYANOTRAP will be a small, natural body of water. And finally, we asked the general public about their opinion on CYANOTRAPs outer design and when we get to build the prototype, we will make it according to the ideas of our respondents.