HUMAN PRACTICES

MEETING WITH PROFESSIONALS

APPLICATIONS

EDUCATION & PUBLIC ENGAGEMENT

https://2018.igem.org/Team:Evry_Paris-Saclay/Public_Engagement

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 [1]. When we met Jérôme Bonnet (link to Human Practices), 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 [2] 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 pLlac 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

https://2018.igem.org/Team:Evry_Paris-Saclay/Collaborations

REFERENCES

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

[2] 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.