HUMAN PRACTICES

Beyond and alongside the scientific project, the human practices are the long story of the project building and the questioning about its applications in society. This essential part of the project allows the production of Biosystems that fit with current society issues considering people needs in a userfriendly and safe way. If the scientific project corresponds to the lab work, then human practices is the link between the lab and the society to gather people around the beautiful world of science and biotechnologies.

"Human Practices is the study of how your work affects the world, and how the world affects your work."

Peter Carr, Director of Judging

MEETING WITH PROFESSIONALS

In the framework of Human Practices, we initially began with meeting professionals in the research community to verify if our foundational advance project: PEPTalk was innovative and if it seemed as useful for the world of research as for the public. Here are the main professionals we interviewed.

Jérôme BONNET
Researcher at UMR 1054 INSERM in Montpellier, FRANCE.

During this interview we discussed about the potential of our project to improve co-cultures in various projects. We had the opportunity to find many applications of our project to different laboratory techniques. See the Laboratory Aspects of the Applications.

Dr. BONNET also recommended other articles from other researchers in the field of logic gates. He advised us on some details that we should consider such as the permeability of the membrane of each bacterium to the viral peptide that serves to communicate, so we checked the presence of the Opp transporter in E. coli (see the Design Overview page).

Overall, he was quite enthusiastic about the project and testified the real usefulness of PEPTalk in research topics using biosensing systems.

Gregory TUFO
Responsible of business (chargé d’affaires) at Genopole in EVRY, FRANCE.

This interview was very interesting. This time we had to meet someone who was not an expert in synthetic biology, who just had some knowledge in microbiology.

We discussed about the potential of our project to interest professionals in both research and industry. Mr. TUFO was also very enthusiastic with a view of adapting our system in the field of therapeutics or biosensing.

He advised us to direct our interest on probiotics acting on the intestinal microbiome and on developing this aspect to show that our project brings something new. He also suggested that we should be interested in the environment by investigating the possibility of a bulk pollution biosensing system in different types of ecosystems.

Overall, Mr. TUFO has helped us greatly enriching our network of contacts in different areas of research, in addition to giving us valuable advice on how to present our project to the professionals we meet.

Volker DÖRING
Researcher at Genoscope CEA in EVRY, FRANCE.

The purpose of this interview was to find out if our project could potentially be useful for an existing research project. That is why we met Mr DÖRING who is in charge of a research team of Genoscope CEA in Evry where they work on a continuous culture machine called GM3, more commonly called “the farm”. It is a large complex system of tubs connected to bottles of specific media that allows cell cultures for months or years and taking a generation for study if necessary. The advantage of this machine is that it can significantly extend the life of any cell culture and store any mutant.

We therefore discussed the potential of our project to adapt to their research tools to open the way for other innovative research projects. Mr DÖRING was very motivated to work with us, he believes that our system can adapt very well to GM3.

He also informed us about the different parameters to consider when we want to work with co-cultures such as the choice of environment, the interactions between different bacteria, the potential conjugations, but also according to the size of the population and the empirical potential of the evolution of our strains, the possibility of mutations that we must not forget to consider.

This interview made us notice that many research teams are interested in a stabilizing co-cultures for their research, something we can bring to them with PEPTalk.

APPLICATIONS

Human Practices highlight the impacts that a project could have on humans and the world around them. These impacts can be economic, social, environmental as well as medical. They can be beneficial or harmful.

Our project, as a foundational advance, aims to transcend disciplines. It has the advantage of being able to affect many applications, specially quorum sensing applications as, for instance, biosensing [1] and distributed logic gates [2]. Of course, depending on the spatio-temporal context, the possible applications of our project may raise ethical and legal issues.

We will, in this part, show that the benefits outweigh the disadvantages which, in turn, can be overcome.

Laboratory aspects

As part of the Human practices project, we were interested in how to apply our system to an existing research project. To do so, we have received valuable advice from researchers in the field of synthetic biology and microbiology.

CO-CULTURES

After our meeting with V. DÖRING, we have wondered if we would be able to apply our PEPTalk system to their GM3 machine.

The small disadvantage we found in this machine is that we can only grow simple cultures, and all research projects using this machine are focused on simple cultures. We are developing a system that is able to make co-cultures possible and stable. So we asked Mr. Volker DÖRING, project manager, and Ivan DUBOIS, GM3 engineer, to tell us if it was possible to link our two tools. The farm team told us that our system can be applicable to GM3.

Besides the fact that we have to take into account a lot of culturing parameters that are quite restrictive when we talk about co-cultures such as the compatibility of our strains in terms of nutrient medium, temperature but also interactions between them. Our system can be a serious asset in the study of mutations. The GM3 is a perfect tool to study these mutations because it allows us to study them over long generation times, monitoring the time of evolution of our modified strains. Mr DÖRING suggested that we focus on the rest of our tests at this point.

If we are able to bring co-cultures to GM3, we are sure to pave the way for many interesting research topics around co-cultures.

LOGIC GATES

During the interview, J. BONNET helped us to find some applications that our PEPTalk system could improve and optimize.

A first application would be to give to each species of bacteria in the coculture a different peptide signal. Therefore, we can adapt a quorum sensing between all the species present in the medium, as well as decide to treat them separately with a little group of peptides.

Because of that advices, We have thought of a way to generate a large variety of peptide / receptor pairs in a future part of the project. See the Future Perspective page.

A second application would be to use our communication system as a signal amplificator. For instance, in a biosensor which detects the presence of a molecule (example: arsenic), the reporter signal can be very weak when the quantity of arsenic is low, getting mixed into the background noise. This is because few bacteria are in contact with the arsenic and produce the reporter. To amplify the signal and discrimin low concentration of arsenic, we could use our peptide as a diffuser of the induction information to other bacteria in the medium (by amplifying the signal of induction).

J. BONNET told us that our communication system could have been very useful as an information diffuser in one of the bacterial biosensor systems that his team used during their research project on logics gates [4].

Economic aspects

From an economic point of view, our system promises to revolutionize and control genetic circuits through the use of hexapeptides as genetic inducers. Using peptides is a cheap and accessible method, unlike the high cost of the IPTG inducer which is more commonly used in this kind of projects [3].

Medical aspects

From a medical side, we can allow more accurate estimates of the diagnosis of a disease. For instance, using a biological biosensor with our system as an signal amplificator would increase the sensitivity and reliability of detecting a malignant tumor in the body. Besides, our system could allows the detection of several factors that interest us at the same time (see the Logic Gates chapter). This will allow the detection of cancers as quickly as possible to hope for an optimal and total cure.

Environmental aspects

From an environmental side, it will be possible, for instance, to detect micro plastics or endocrine disruptors with much more reliability than before. Indeed, our project, once again would detect a wide variety of bio component via a biosensor based on a logic gate with multiple inputs. This signal would be more sensitive than existing tools thanks to the broadcast system. As a result, we could better understand the pollution problems we are facing, from a macroscopic as well as a microscopic point of view. Because the pollution we can’t see is as important as the one we can see.

Legal and ethical aspects

Our project aims to improve several areas by introducing a biological aspect. Often, using bacteria as a medicine can be very controversial depending on the country. Even if people can ask ethical questions about the use of our communicating bacteria, it is surmountable. Let's take an example of an application that could pose a problem:

In the same way as a probiotic, our communication system would be useful for regulating the human intestinal microbiome and preventing the spread of infectious, pathogenic bacteria.

The idea would be to integrate our system into a bacteria which is already present in the body. However, it will be "a genetically modified organism". Thus, GMOs raise ethical issues about health and the environment.

Indeed, since the seasonal cycle in the human microbiome does not necessarily generate a total suppression of all the enzymes, the new genetically modified bacterium could never completely die and thus remain permanently in the human gut.

Then, from an environmental point of view, there is the question of genetically modified bacterium that humans can reject and that could be harmful to the outside world. The current European and national laws allow to limit the damage thanks to a legal framework. For example, the European law, in the directive number 2001/18 / EC of the 03/12/2001, concerning the deliberate release of genetically modified organisms into the environment, that states the principle of precaution.

Also, our "new bacteria" created in capsule form will have to comply with marketing authorization laws as well as the principles of therapeutic necessity. Our genetically modified bacterium, is concerned by the drug definition of the French law. That requires to respect the step for marketing authorizations, according to the article L5111-1 , 1st and 2nd paragraphs, of the French code of the public health.

In any case, our system can be adapted without worries to a legal and ethical probiotic. It will also be more interesting than other existing probiotics because it will prevent an infection even before being confronted in a sensitive way and in long-term. An indispensable tool for travellers visiting a foreign country.

In general, our project would contribute to the improvement of the quality of life, health capital, and well-being capital. In addition, the PEPTalk tends to adapt to different areas of application, and this, in compliance with ethical and legal laws.

Improving previous iGEM projects

In this part, we will focus on the old iGEM projects led by our colleagues. The goal is to show how our PEPTalk system can improve or change some parts of previous iGEM projects.

IGEM TOULOUSE 2013

This very innovative project tended to create an electronic system called "E.calculus" which serves as a "bit calculator". The main goal was to demonstrate that a system based on recombination logic gates could be used in complex systems. Thus, the bacterium that received this system would be able to make its own calculations and send a report to the user.

E.calculus consists of 5 logic gates: 2 XOR, 2 AND, 1 OR. The general idea was to create a complete and stable complex system in a single bacterial strain. The logic gate would be regulated by "tetR". In the absence of an inducer aTc, tetR would be produced and would repress the expression of the entire logic gate system, allowing the bacterium to develop normally. On the other hand, adding the inducer aTc would move the bacteria from a "normal" state to a "calculator" state (see the picture).

Because the system is too large to fit into a single plasmid, they decided to integrate the logic gate parts (AND1-AND2 and XOR1-XOR2) directly into E. coli DNA (see the picture).

Where our project can improve this old project is at the level of logic gates. We know that a plasmid cannot accept too many bio bricks. That's why they took the initiative to cut the system and integrate it into the microbial DNA. The problem with chromosomal DNA is that you can only have one copy per bacterium while the plasmids can be present in several copy. We could just insert several plasmids but there is a potential problem of rejects and the bacterium would die because it would not have had enough energy to support all the plasmids.

Our system avoids all these problems. Indeed, thanks to PEPTALK we can insert each logic gate in a plasmid which will also be inserted in a different strain of bacteria. The communication system would simply connect the different bacteria and therefore the different parts of the E.calculus system to increase its efficiency and give positive and non-random in vivo results. This is called a distributed logic gate.

IGEM LUND 2017

This team had the wonderful project of creating a biosensor detecting micro plastics in water. This project is based on the creation of an AND-Gate (see the picture). with phthalates and organic pollutants as input, and a protractor as output. The system is activated in the presence of IPTG.

This project is indeed very interesting because it involves biosensing using logic gates but the number of components it can detect is very small.

Thanks to our PETalk system, we could easily and significantly increase the number of detectable components via a wider variety of receivers connected to a large number of communicating doors. This would expand the field of bio pollutants to clean up.

EDUCATION & PUBLIC ENGAGEMENT

While in France the general public and even the first university cycle biologists decrease Biology to the fauna and flora observation, we tried to speak to a large target, initiated to the synthetic biology or layed. Our aim was to get known synthetic biology and its severals possibilities. Click here to see our Education and Public Engagement projects.

INTEGRATED HUMAN PRACTICES

During our Human Practices research, we quickly realised two things. First, there is a real and pressing need for new bacterial communication systems for researchers. According to this assessment, we have investigated in detail what kind of communication system would fit the most to current research needs and interests. Second, the general public has limited knowledge about biology, which makes it really hard for them to understand a foundational advance project such as ours. Therefore, we decided to make great efforts in educating people about biology and synthetic biology.

Choosing the communication system

Bacteria communicate through quorum sensing (QS) systems that can be classified into two kinds based on the signalling molecule used: small molecule (AHL) based QS and peptide based QS [5]. When we met Jérôme Bonnet, he explained to us that it would be very useful to characterise a number of different orthogonal communication molecules. Since peptides are easier to engineer than small molecules, we decided to focus on a peptide based QS. After we had engineered the system for our chassis of choice (E. coli), we expected to be able to generate several variants of it through directed evolution.

In addition, our communication system had to be as orthogonal as possible to avoid crosstalk with other variants of this communication system as well as with other natural communication systems inside the cell. Therefore, we found the “Arbitrium” bacteriophage QS [6] to be a good candidate, since the orthogonality would be better using a bacteriophage system non-native to our bacterium.

Finally, in the native “Arbitrium” system we have a promoter that is activated by an activator protein, which in turn is deactivated by a small signalling peptide. Since the activator is expressed constitutively, the overall system behaves like a constitutive promoter that can be inhibited by a peptide signal. This kind of promoter is very useful for logic gate constructions. However, many labs and companies are used to working with promoters that are activatable by an inducer molecule, such as pLacI by IPTG, pBad by arabinose, pLtet by tetracycline and pLasR by acyl-homoserine-lactone. Therefore, we investigated whether our system can be switched from a repressible promoter to an activatable promoter, using the original promoter as an operator sequence downstream of a constitutive E. coli promoter. Consequently, we developed a promoter which responds to a constitutively expressed repressor and a “de-repressor” peptide.

Choosing the chassis

After we identified the “Arbitrium” system, a decision had to be made: which chassis should we use? We initially thought of using B. subtilis, which is the host of the SPbeta bacteriophage group, to characterise a library of orthogonal QS signals. However, our Human Practices research revealed that most labs and companies are working with E. coli. So, we decided to adapt the “Arbitrium” system to fit the E. coli chassis, which as a Gram-negative bacterium does not naturally use a peptide based QS. Once we are able to establish a peptide based QS in E. coli, the characterisation of a library of new QS peptides would become possible.

Popularising the project

During our Human Practices research, we realised that it was not easy to explain our project to the general public and make people interested in it because it was very abstract for them. So we decided to do two things. First, we investigated a number of potential applications arising out of our project (link to Applications), such as gene regulation, distributed logic gates, and multiplexed biosensors. Next, we have created many different media to educate the public about each of these applications. The orthogonality of a communication system is explained through our video game (link), distributed logic gates are explained through our interactive rug (link) and other applications were described during the many events we participated in (link).

COLLABORATIONS

During this iGEM season, we have met amazing people from all around the world and performed great work with some of them. Click here to see our collaborations.

REFERENCES

[1] Choudhary S, Schmidt-Dannert C. Applications of quorum sensing in biotechnology. Appl Microbiol Biotechnol (2010) 86, 1267-1279.

[2] Kylilis N, Tuza ZA, Stan GB, Polizzi KM. Tools for engineering coordinated system behaviour in synthetic microbial consortia. Nat Commun (2018) 9, 2677.

[3] Hennig S, Rödel G, Ostermann K. Artificial cell-cell communication as an emerging tool in synthetic biology applications. J Biol Eng (2015) 9, 13.

[4] Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science (2013) 340, 599-603.

[5] Federle MJ, Bassler BL. Interspecies communication in bacteria. J Clin Invest (2003) 112, 1291-1299.

[6] Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. Communication between viruses guides lysis-lysogeny decisions. Nature (2017) 541, 488-493.