Team:INSA-UPS France/Description


Combine to improve: a synthetic biology scale-up

Nature offers us a wide variety of tools in order to sense precise chemical or physical parameters. For example, enzymes and receptors are specialized for unique or restrained range of molecule, or even for specific light wavelength.1,2,3 Obviously, as mankind progressed in the comprehension of life science, he wanted to take advantage from this extreme diversity and specialization. Synthetic biology was born thanks to those expectations in the early of the twentieth century4 and made quite notable breakthrough. However, most of those works were based on the insertion of a lot of DNA information on one single model, typically E. coli. We observed that all those informations are not always compatible with the use of a unique microbial chassis : although standard microbial models are quite modular, they have a particular membrane, specific pathways and different ways to do protein maturation.

Why should we persist to use a single model when we have access to a wide diversity of organisms?

For our iGEM project, we decided to focus on the multi organisms aspect, making communication between prokaryotic and eukaryotic possible.

A core: prokaryotic-eukaryotic communication

Our first challenge was to design an eukaryote-prokaryote communication system, so we can have a standardized way of communication, with modulable input and output. This system answers to some of the synthetic biology current challenges8 that are: device modularization and standardisation with the core, using the diversity of living systems and so, using the right cellular chassis, with the right genetics elements.

Team SCUT26 already described a binding-receptor system built on Saccharomyces cerevisiae18, based on the Odr-10 receptor, a G Protein Coupled Receptor isolated from Caenorhabditis elegans19. The pathway is engaged by the activation of Odr-10 and has been shown to start the mating cascade which ends with the activation of the pFUS1 promoter by Ste1220. After a discussion with Diethard Mattanovich , the supervisor of iGEM Team Boku Vienna, it appears from his transcriptomic data that the mating pathway including Ste12 was also present on Pichia pastoris, a yeast gaining more and more interest for being a robust tool for protein and metabolite production.33 Once we found a molecule factory, we needed to find a way to induce it, whenever the bacteria sensor we were using. Checking the metabolism of diacetyl on KEGG Pathway, we identified that a simple enzyme, the acetolactate synthase (ALS), processes to produce the diacetyl from pyruvate, a ubiquitous metabolite.

As this mechanism relies on diacetyl detection for pFUS1 activation, we needed the sensing module to produce this molecule. Online tools predict that the missing enzyme to catalyse the production of this molecule is the acetolactate synthase (ALS). Indeed, this enzyme catalyses acetolactate production, which is then oxidized into diacetyl. Thus we decided to add the gene responsible for ALS production in the genetic construction we put in the sensing module.

A microbial consortium against cholera

Recently, research teams started to focus on synthetic biology in order to find a way to deal with Vibrio cholerae5,6. Additionally, some iGEM teams also took the challenge of detecting V. cholerae, using E. coli as a host for the quorum sensing system, but it seems that this strategy did not succeed7 or showed mixed results8. Most of the iGEM teams intended to prevent the cholera infection thanks to gene targeting such as cleaving toxicity genes9 or inhibiting specific genes on the pathogen10. With this lack of successful stories in iGEM on mono-microorganism and the need to have a whole specialised lab in order to build a complex system of synthetic biology, this statement could be perfect to test our core in real conditions and with a useful application.

Finding a sensor

First we had to detect the initial state of the system in order to activate the core. An interesting property of Vibrio cholerae is its quorum sensing system that depends on the CAI-1 molecule. CAI-1 directly reflects the amount of V. cholerae on water, and thus its virulence. Once CAI-1 is at high concentration, it can be detected and then provides us a good way of detecting V. cholerae once it becomes pathogenic19. Vibrio harveyi, a non-pathogenic strain of Vibrio, showed itself as a perfect sensor of the CAI-1 molecule and thus of the quantity of V. cholerae. This strain fulfilled the requirements we wanted as a good synthetic biology chassis: easy to engineer, already has part of our final system such as the CqsS/CAI-1 pathway19,20. We only had to mutate the receptor and integrate the als gene to trigger diacetyl production in presence of CAI-1.

Quorum sensing mechanism in V. cholerae

For safety reasons, we were not able to manipulate V. cholerae in our lab. This is why we had to engineer E. coli to mimick V. cholerae in order to further test our system. The idea was to make E. coli produce the specific quorum sensing molecules of V. cholerae: CAI-1.

Finding an effector

Because of our will to kill gram-negative bacteria, we assessed that a prokaryotic chassis could not be adequate to achieve the desired effect without drawbacks. So the information of V. cholerae detection must be transmitted to an eukaryotic protein expressor : Pichia pastoris. Once we had both the sensor, and the core, we had to act on the system. In our application, we aim to reduce the amount of V. cholerae, or even better, to totally kill it. We found the solution thanks to an unorthogonal approach: we looked in the environment of V. cholerae in order to find out a competitor, or an organism not affected by it. It appears quite fast that crocodiles have an impressive immune system, that allows them to resist against lots of diseases vectors infections22,23,24. We used from their amazing immune system promising molecules: antimicrobial peptides that have proven to have a good killing efficiency against V. cholerae13,14. Moreover, this effector, with a good efficiency against bacteria and the need to be secreted, was a perfect effector to implement in our multi organisms system with specialised tasks.

Those peptides are cationic molecules and can target bacterium membranes, to create pores in it, leading to the lysis of the cells24.

The fact that literature described the robustness of Pichia pastoris16,17 for the production of antimicrobial peptide made us definitively enthousiast about using this yeast as part of the core.

Our system