Team:Montpellier/Human Practices

Integrated HP

The Project with the Public

Public’s opinion

During our project we had the occasion to talk about our research to a lot of different people (more information can be found in our Education and Public Engagement page). Those discussions were informative about the view people could have about synthetic biology and our project. Some of the main concerns can be found on Figure 1.

Figure 1: Concerns and comments people gave about our project.

Survey

By having the idea of our project, we immediately realised that we needed a way to introduce our project to the non scientific community. Moreover, in order to integrate our project into society, we first need to know what the people want, and also, how deeply they know the subject.That’s why we first created a survey asking questions about contraception, hormonal treatments, the use of genetically engineered organisms and consequences on the environment.

We wanted to have an overview of the perception of contraception and genetically engineered organisms by the general population. We developed a poll to probe as much people as we could: our survey was answered by 1163 people in both English and French. In the poll we are asking general questions about people, their contraceptives mean, their satisfaction about it. Then we went further with question about GMOs and their perceptions, then asking their view about a contraceptive mean such as ours.

The survey was mainly answered by women (Figure 2).

Figure 2: Pie chart of the Gender Identity.

The people that answered the poll were fairly young with an over representation of people between 18 and 25, over 50% of the poll responders (Figure 3).

We also have an over representation of people with high level of education (Figure 4).

Figure 4: Pie chart of the education level.

And finally nearly all probed people were from France (Figure 5).

Then knowing our population sample we asked their contraceptive mean (Figure 6).

Figure 6: Pie chart of the contraception method used.

This was more or less representative of the general population of France, except for the so called “natural method”. We discovered that militants of this method heard about our project, and decided to make their voice heard about how they disprove it. We wanted to notify this only because they were over represented in our poll (about 20%) compared to the French population (3%) and hence could biased our poll. As a note the “natural method” is based on temperature measurements and self-assessment of the viscosity of vaginal secretions. However it seems relevant for us to mention that seems can not be seen as a contraception mean and that it is not as efficient as contraception methods. Natural methods use represent 21.8% of unplanned pregnancies cases which is the higher rate for any contraception method [12].

Contraception Satisfaction

We then asked the amount of satisfaction on their contraceptive means on a scale from 1 to 5 (giving a median of 3), the general average was of 3.74 out of 5 wish reflect a good overall satisfaction. But they were some disparities. We studied especially the most common ones: male condoms and pills. For condoms the satisfaction was of 3.34, which is quite high compared to other method. Whereas for the pill it was 2.26. Showing that the pill isn’t a very satisfactory contraception method. Furthermore 20% of the women using pills or other hormonal means have some form of medical issues or contraindication. So there is actually a place for some new non-hormonal contraceptive mean such as our project.

Impacts of Hormones

After that we asked if they think hormones had impact on the body and our environments. 97% believed it has an impact on the body, but only 85% agreed it had an impact on the environment. We studied those other 15% and discovered they had a lower educational level than the rest of the population, suggesting their belief resulted on a lack of access to information.

GMOs Opinion

We also asked about general view on GMO, and 50% of people think it is dangerous for health and the environment, 18% think it is a useful process, and the rest agreed it depends on the applications and the genes implanted. By isolating data of the younger part audience (18-21) we discovered that they were more inclined to think it is a useful progress 26% (+9 points). However this showed that half of the French population still believes that modified organisms are harmful not matter their application. This was quite a surprise to us because, even though we knew are country to be historically opposed to the use of GMOs we didn’t think the proportion was still this high in 2018.

The Project

Finally we wanted to see the perception of our project from 1 to 5 again, and it turned out it was around 2.95, just above average which is quite reasonable as it’s a bit controversial to propose a contraceptive mean using GMOs. We concluded there is a place for a new non hormonal contraceptive mean for a part of the population but that still a majority was probably not really aware of what modified organisms were and how they could serve their health and environment in many different ways.

Conclusion

This polled changed our view of how we should talk about our project, knowing how bacteria and their microbiome was a prerequisite for people before they could understand the extents of our project. This is why we decided to bring something to the science popularization movement. We wanted to create something that could explain to people how the female reproductive systems work, how contraceptives method do but also what synthetic biology is and present our project. We conducted different projects to fulfill this goal that we are presenting in the following sections.

Interaction with professional gynecologists

During our journey, we were able to discuss our project with a few french Gynecologists having different points of view about our project:

Elisabeth Paganelli, Tours
Claire-Noëlle Collardey, Paris
Olivier Cherici, Montpellier
Laurent Vandenbrouck, Montpellier

According to them, the scientific world knows well about probiotics, and their use is not an innovation. What is an innovation is to use genetically engineered lactobacillus as a probiotic. It is also necessary to turn to non-hormonal contraceptives, since people tend not to want it anymore says Dr. Elisabeth Paganelli.

However, the use of such bacteria raises different problematics such as potential noxious effects for the vaginal flora and the vagina. The use of the Antimicrobial Peptides coupled to Antisperm Antibodies could increase vaginal irritations and therefore rising the chances to get a Bacterial Vaginosis.

In addition to that, people's vision toward GMO is quite bad (and especially in France) “That's why a product like Vagineering should get a very strong marketing and brand associated” said Paganelli. And the fact that the product is, obviously, reversible should be made very clear from the beginning.

According to Dr. Laurent Vandenbrouck, the vaginal microbiota renew itself around every 3 months, so there is a necessity to find an adequate gallenic formula.

All gynecologists agreed on the fact that the world needs new contraceptives and that the scientific community should work on new alternatives to hormonal contraceptives.

In conclusion, we highlighted a lack of knowledge and research around the vaginal flora. For a project such as Vagineering to really exist and be proposed, it is essential that non-scientific people better understand what is the microbiota and synthetic biology.

In addition, there is a lot of concern about our project. Indeed, there is a fear of a problem of reversibility and spread of bacteria. However, the project is largely supported, showing the interest of new ways of contraception.

Popularization

As it is probably one of the most important aspect of our project, since it directly applies to the Public life style, we decided to create different tools for the people to understand better, and make their own point of view of our project.

How? By interviewing them, knowing what could be their questions. Educate them about the vagina and its flora, contraceptives and everything about it, and synthetic biology and its possible application for women health.

A Comic Book: Integrate our project into society

As you saw earlier, more than 1000 people answered to our survey. The idea is that following the survey, people can give feedback and ask questions. So, we wanted a way to answer to their questions in an educative way.

With the help of the artist @Minskiga, we created a popularization comic book.
This comic book has 2 main objectives:

The first one is to answer to the community questions about contraceptives and synthetic biology applied to it.
The second one i to create a path between scientific knowledge and a lay audience through a storyline.

We also want our comic book to be accessible for every level of education starting from 12 years old.

Steps of creation

Storyline: who will tell the story? What questions to answer?
Bibliography: find the papers that talk about our subject and go deeper and wider.
Drafting a storyboard: the human practices team drew all the storyline.

Tamara's first drawing

Minskiga's final drawing

First correction: English spelling and scientific corrections. We also discussed the point of view of our comic book.
Artist work: We then sent the storyboard to Minskiga, and she started her hard work !
Finding a Name

The relationship between us and the artist

Minskiga is an artist student who hasn’t any biology education and therefore will have her own perception of scientific words.

Since she is living in a different place than us, we saw her only a few times during the summer.

It is necessary for the artist to well understand the subject in order to popularize it.

As you can see in this example, Minskiga first understood that Lactic acids were producing bacteria in the teen vagina.

However, this is the exact contrary : Lactic bacteria produce lactic acids.

⇒ We had to discuss about exactitude of her drawings, and we still are modifying pages.

The relationship between the artist and the public

Minskiga uses her art skill to describe something that some non educated person will not obviously understand through scientific words. And she give an illustrative answer to a question. It’s actually reading a comic book instead of reading a scientific paper.

We created characters for the public to identify themselves in the story.


Minskiga	Vulva	Bacterium
She’s the Artist that creates the comic book. She met a team of young scientists and she wants to share their knowledge	She’s minskiga’s friend and she knows a lot about vulva anatomy and microbiota	It’s one of the bacteria living inside a microbiota !

Answering questions from the survey and social networks feedback through the comic book

Through social networks and our survey, people asked us a lot of questions about our project. We used those questions to build a storyline and answer to most of them.

What is a vaginal flora?

We described the concept of microbiota. Minskiga explains of what it is constituted, and where it is. This page also present differents properties of a microbiota and especially for the vaginal microbiota.

What is your sexual identity?

Here, we give a possible definition of sexes and gender identity.

Someone asked for more education about genetically engineered organisms

We decided to create pages to educate people to synthetic biology by giving a concrete example.

Bibliography

For each and every pages of the comic book, we had to search and confirm informations in scientific papers. As we will provide knowledge to non educated people, we need to have sources for everything we said.

However, our comic book is made for the people to start a reflexion about contraception and synthetic biology. The knowledge we give is therefore not exhaustive.

Page	Information	Reference
5	Vaginal microbiota	Huang, B., Fettweis, J. M., Brooks, J. P., Jefferson, K. K., & Buck, G. A. (2014). The changing landscape of the vaginal microbiome. Clinics in laboratory medicine, 34(4), 747-761.
9-10	Environment affecting the microbiota	Fashemi, B., Delaney, M. L., Onderdonk, A. B., & Fichorova, R. N. (2013). Effects of feminine hygiene products on the vaginal mucosal biome. Microbial ecology in health and disease, 24(1), 19703.
11-13	Infectious diseases	Couic-Marinier, F., & Pillon, F. (2017). Prise en charge d’une mycose vaginale. Actualités Pharmaceutiques, 56(568), 14-16. Petrova, M. I., van den Broek, M., Balzarini, J., Vanderleyden, J., & Lebeer, S. (2013). Vaginal microbiota and its role in HIV transmission and infection. FEMS microbiology reviews, 37(5), 762-792.

Conclusion

This comic book is the result of more than 3 months of work, and still needs to be improved. Therefore pages will being modified again before its release. But it will be uploaded very soon. You can find out more about this project in our Public Engagement page !

What’s next?

We want to edit and publish this comic book. Indeed, Minskiga did, and is still doing a very hard work on the project, and we are proud of it. So, after the presentation at the Giant Jamboree, we will try to print the comic and license it.

Biosafety

Working within the thematic of contraception raises many questions: sterilization, contamination, pollution... We will discuss these points below.

Antibodies Risk

Among our spermicidal molecules, we used the antibody YLP20 and the antingen YLP12 that can cause infertility in women and men. That is a question that we had through our project : can you create a virus? Maybe the use of YLP12 can trigger a sterilization of the user? It must be known that the YLP12 antigen is already present in the human body. To be fertile, there must be an autoimmune response. As we have summarized, YLP12 has no immunogenic power.

The creation of a contraception vaccine (CV) is a reflection that has been taking place for quite some time. For example, animals [1] and human [2] studies have shown that deliberate immunization of males and females with sperm causes contraception effect. So, with recent advances in the fields of vaccinology, adjuvants, and nanotechnology it should be possible to realise a CV [3].

However, if we can create a CV, we can create a virus. The limit is very fine between virus and vaccine. Indeed, a vaccine must not itself cause illness or death, it should be protective and gives sustained protection. It induces neutralizing antibody and protective T cells.

In conclusion, there is no risk at our level to create an immune reaction and thus no risk of sterilizing the people because of this antigen.

Dissemination

Working with organisms aimed at vaginal flora implies a very thorough reflection on the spread. The people we interacted with showed a lot of reluctance to our project because of the following points concerning dissemination:

in the environment
inside the body
between people
from mother to child
to a man flora

That’s why we thought at several biocontainment strategies according to prevent unintended proliferation of GMOs in natural ecosystems to chronological time.

Physical containment

If we would like our product to be distributed in a few years, one of the best answer is probably to use a physical containment. The membrane’s nanoporosity allows spermicidal molecules produced by L.jensenii to pass through the membrane, but the bacteria remains confined.

For that, we can use biocompatible polymers. A possible biocompatible polymer could be bacterial celluloser.

We could imagine a design of cellulose Whatman like filter maintaining our modified L. jensenii inside the container and spermicidal molecules to be able to pass through the filter (Figure 7).

Figure 7: Design of the filter for physical containment.

During one of our meeting with the iGEM Toulouse team (you can check our Collaborations page), we discussed about the dissemination of modified L. jensenii in our system.
As their bacteria can produce cellulose, we thought we could build a containment system made of bacterial cellulose in order to confine the cells.

However, this system would probably make the interval of intake shorter due to the probable death of the confined bacteria. What is positive is that system is fast, effective, reversible and avoid interaction between our bacteria and the commensal microbiota. However, such a system is similar to Intrauterine devices as it is still invasive. In addition, and we do not stop horizontal transfers.

Kill switch

Toxin antixin avoiding mutation

Deadman circuit uses a small molecule binding transcription factor to produce a “survival” state in which repression of toxin production is linked to the presence of a specific environmental signal.
Upon loss of the environmental signal, the circuit switches to the “death” state in which de-repressed toxin production kills the cell [4].

Temperature kill-switch: avoid dissemination

Cyodeath utilize a toxin–antitoxin system in which toxin CcdB is expressed if optimal temperatures are not met. If the memory element is lost and the transcription factor is absent, the essentializer system induces production of CcdB and the cell dies [5]. Thus, our engineered Lactobacillus jensenii would live inside the body (37°C) and, outside of the body, the bacteria would die.

Organisms engineered for synthetic auxotrophies

Imagine that you drink your coffee each morning and this permit your contraception to work properly! And when you stop, your dependant-caffeine bacteria die. This kind of kill-switch could be a good way to sustain survival or trigger destruction of engineered cells using molecules not present in the body and widely used by humans!

The C-STAR system developed by Bojar et al. can [6] induce gene expression with caffeine by enabling engineered mammalian cells to directly sense caffeine at physiologically relevant concentrations. Thereby, it’s making possible to fine-tune therapeutic transgene expression in response to routine intake of beverages, such as tea and coffee without supplementation of any additional chemicals! In this paper, cells are modified and it express expression pathway different that we want. We are thinking about this system for our bacteria. Indeed, a system exists in E.coli that react to caffeine. This is the case of a caffeine sensor, developed by our advisor Hung-Ju Chang in Montpellier [7].

A similar strategy is to design organisms with an altered chemical language that isolates them from natural ecosystems. The idea is to redesign essential enzymes in an organism possessing an altered genetic code to confer metabolic dependence on non-standard amino acids for survival. Mandell et al. assigned the UAG stop codon to incorporate a non-standard amino acid (NSAA) and computationally redesigned the cores of essential enzymes to require the NSAA for proper translation [8]. Thanks to that, they show that organisms exhibit unprecedented resistance to evolutionary escape through mutagenesis and horizontal gene transfers!

Moreover, the bacteria can have a synthetic amino acid [9] or a synthetic nucleotid and their absence is lethal.

Switch-off

At the end, the best solution is to keep L.jensenii in our microbiota all the time and make it produce or not the spermicidal molecule! Challenges remain before these engineered bacteria can meet the market. For that, we met different problematics. We need a better understanding of how the bacteria interact with the body and other organisms.

Switch-off

First of all the concept of turning ON/OFF a gene is well studied. Many mechanisms allow the extinction of a gene. However, we must add a "memory" in the DNA of bacteria to maintain the repression.

HiFi Genome

A genome is bound to undergo changes trough single nucleotide polymorphisms (SNPs) and other environmental modifications. The thing is, in our project, the action of epigenetics and SNPs aren’t welcome.

That’s why we need to maintain the genome integrity. We need to take care of editing the bacterium's chromosome, rather than its plasmids because is it too “mobile”. We need to limit the bacteria ability to share DNA with other bacteria which would create long-term safety risks [10].
Gallagher et al. [11] employed safeguards based on auxotrophy, independent essential gene riboregulation, repressor supplementation, and engineered addiction to eliminate persistence and limit escape frequencies. This work describes the implementation and advantages of multilayered genetic safeguards in E. coli, so we can imagine a same system in L. jensenii.

Moreover, the bacteria can have an amino acid synthetic [9] or a synthetic nucleotide to create a synthetic organism that can not interact with the other bacteria from the microbiota. So, it is a possibility to create synthetic nucleic acids.

Conclusion

Through our new method of contraception we encountered a lot people doubt because of the safety of the project. On the one hand, those doubts are linked to the lack of knowledge in synthetic biology and the vaginal microbiota but it is also linked to the limitations of current technologies.

To answer these problems, we focused our efforts on the popularization of the subject through arts, events, and our comic strip. But we also thought about several ways to limit the contamination and dissemination of the strain to tend to a realisable project.

However, our project is a possible application with vaginal microbiota in synthetic biology. We do not pretend to create a new way of contraception in the years to come. It is not a new mean of contraception close to be on the market: for now, our project consists of having spermicidal molecules produced by a bacterium. During our project, we ignored pharmaceutical aspects (doses, side effects, safety in the organism, posology etc. )of this kind of device, this study is too new for having that kind of consideration. This kind of aspects would be take in count once we will have our bacteria well transformed and all the control devices performed.

Making a contraceptive is a way to exploit the potential of the vaginal microbiota. The aim of the project is to better characterize L. Jensenii, to make it a new chassis for synthetic biology and to show the draft of a possible application of its use by opening doors to either new contraceptives or to ways to reduce infections to STIs and thus their transmission. All those reflections lead us to the different perspectives we thought about, that are present on our Perspectives page.

References
[1]	Baskin, M. J. (1932). Temporary sterilization by the injection of human spermatozoa. A preliminary report. American Journal of Obstetrics & Gynecology, 24(6), 892-897.
[2]	Mancini, R. E., Andrada, J. A., Saraceni, D., Bachmann, A. E., Lavieri, J. C., & Nemirovsky, M. (1965). Immunological and testicular response in man sensitized with human testicular homogenate. The Journal of Clinical Endocrinology & Metabolism, 25(7), 859-875.
[3]	Rajesh K. Naz. (2014). Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide. Protein Science 2014 Vol.23:857—868.
[4]	Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman'and'Passcode'microbial kill switches for bacterial containment. Nature chemical biology, 12(2), 82.
[5]	Stirling, F., Bitzan, L., O’Keefe, S., Redfield, E., Oliver, J. W., Way, J., & Silver, P. A. (2017). Rational design of evolutionarily stable microbial kill switches. Molecular cell, 68(4), 686-697.
[6]	Bojar, D., Scheller, L., Charpin-El Hamri, G., Xie, M., & Fussenegger, M. (2018). Caffeine-inducible gene switches controlling experimental diabetes. Nature Communications, 9(1), 2318.
[7]	Chang, H. J., Mayonove, P., Zavala, A., De Visch, A., Minard, P., Cohen-Gonsaud, M., & Bonnet, J. (2017). A modular receptor platform to expand the sensing repertoire of bacteria. ACS synthetic biology, 7(1), 166-175.
[8]	Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., ... & Church, G. M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518(7537), 55.
[9]	Rovner, A. J., Haimovich, A. D., Katz, S. R., Li, Z., Grome, M. W., Gassaway, B. M., ... & Isaacs, F. J. (2015). Recoded organisms engineered to depend on synthetic amino acids. Nature, 518(7537), 89.
[10]	Attar, N. (2016). Microbiome: Microbial mobilomes differ between societies. Nature Reviews Microbiology, 14(9), 546.
[11]	Gallagher, R. R., Patel, J. R., Interiano, A. L., Rovner, A. J., & Isaacs, F. J. (2015). Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic acids research, 43(3), 1945-1954.
[12]	Bajos, N., Leridon, H., Goulard, H., Oustry, P., & Job‐Spira, N. (2003). Contraception: from accessibility to efficiency. Human Reproduction, 18(5), 994-999.