Applied design: a demarche to bring synthetic biology to real life

Summary

Several iGEM teams have tried to tackle the cholera problem, but none succeed so far. So, we decided to try something different: creating a synthetic microbiota to detect and eliminate Vibrio cholera in drinkable water. We modified three different organisms in the course of this project and the results are very promising. We managed to established synthetic communication between Escherichia coli and Vibrio harveyi. We also successfully engineered the yeast Pichia pastoris to produce efficient antimicrobial peptides from crocodile!

And this was not the end of it. We thoroughly investigated how our innovative solution will be used in the next future. We designed a device with advices from NGOs, professionals and the general public. Safety, ethics, delivery and lifecycle constraints have been addressed in an exhaustive Scope Statement. iGEM is only the starting point of this project since the SunWaterLife Company has already mentioned its interest to develop our system.

1. Solve a real world problem: Cholera is still a widespread disease…

Gathering knowledge about cholera

Cholera is a worldwide diarrheal disease, caused by the ingestion of V. cholera in contaminated water. Nowadays, cholera is still occurring in developing countries, war zones and natural disasters zones.

Identifying a problem with a graphical method

Drinking water shortages and the lack of hygienic facilities in developing countries are the main reasons explaining current outbreaks.

The WHO reported over 1 million cases over the year 2015 and the mortality was around 1%¹. In April 2017, a cholera epidemic burst occurred in Yemen. In August, more than 500,000 cases have already been identified. This epidemic, which turned into a sanitarian crisis, is still surpassing the abilities of the non-governmental organizations to help populations2 in the long-term. This fact highlight the limitations of the current solutions.

Using the first bibliography work, a problem tree was built to analyze the issue of cholera epidemic and confirm our choice to work with antimicrobial peptide to clean water.

Analyzing the current solutions

Therapeutics

The most used treatment is the Oral Rehydratation Solution (ORS), composed of salts and glucose in order to fight the extreme loss of water due to cholera. It can be drunk or injected intravenously depending on the patient and his symptoms4. This curative method does not wipe out the infection.

The therapeutic solutions do not eradicate the disease vector. During treatment, the human is still considered as a V. cholerea reservoir and can contribute to spread the disease. Moreover, all humans resulting fluids have to be considered as Biosafety 2 contaminated wastes.

Prevention

Even if the ORS treatment is very efficient, it would be more appropriate for people to use prevention methods. While direct preventive methods such as vaccination are currently used, they have been shown to have low efficiency3. Efficient scientific detection or purification methods exist, but they require trained personnel and sophisticated equipment which are generally not available in all areas of the world3.

Cheaper and more accessible alternatives consist in attacking the cholera problem at its root by proceeding to water treatment. The current most efficient ways to eradicate cholera from water is sodium hypochlorite treatment. According to MSF, bleach treatment requires dedicated and well trained human resources and a logistic to ensure a good supply. The bleach water treatment is complex: before treating the water the chlorine demand has to evaluated in order to determine the right amount of bleach to treat the volume of water.
Low pH, high temperature, sunlight exposure, metal and turbidity impact bleach stability and correct treatment.¹
Besides, bleach impacts the organoleptic properties of the water, hence presents an acceptability problem. However, according to the WHO (2014), the bleach rendered services are higher than their prejudice.

Another solution consists in filtering water. This prevention methods could be expensive and difficult to set up. Moreover, people living in remote villages do not have easy access to these systems and it can take days to reach a camp to be cured.

The last classic solution is to boil the water, but this could be complicated for big water volumes, especially to maintain low level of V. cholera agent if the water stock is again contaminated through people utilization. Thus, new methods of prevention and treatment have to be developed.

That’s why our system was designed to treat water in most situations. By improving prevention methods and water treatment, we aim to act in favor of universal access to drinkable water based on frugal innovation integrated in local dispositive.

2…in the most elegant way: a microbial consortium

Analyzing the current solutions helped us to take an original positioning for our solution and focus more on the prevention approach by purifying water than on a diagnosis system. Our system is energy free, has a soft impact on the user by being easy to implementminimal impact on organoleptic characteristic of the water.

We were not the first iGEM team to engage in the cholera fight. The main challenge of previous iGEMers was about V. cholerea quorum sensing detection. They worked with E. coli as a chassis but all faced major issues to detect V. cholerea quorum sensing since transposition of signaling pathway between microorganisms is a complex task, even harder if we want to integer a bacterial pathway in a yeast. That was our main reason to choose the cholera problematic to demonstrate the potent of synthetic consortium.

Conditional expression of non-conventional antimicrobial peptides.

First, D-NY15 was shown to be efficient to eliminate V. cholerea ( see Results). Our solution is intended to treat large volume of water. Considering the growing prevalence of antibiotic resistant bacterial strains, we feel that using such a is a great asset to the project. This is reinforced by the expression conditional to V. cholerea presence. D-NY15 use will also be facilitated by the fact that it does not show any cytotoxicity effect on human cells.² ”>

Advantages of the consortium solution

Building a synthetic microbial consortium with V. harvei and Pichia pastoris was a major disrupting decision regarding the current solutions and the previous iGEM works. Eventually, this consortium could be superior to the sum of both single organism. With minimal modifications, Vibrio harveyi can detect V. cholerea but cannot produce AMP because of self-lysis issues. Pichia pastoris cannot directly detect V. cholerea but can produce efficient molecules to eradicate it without self-lysis. The consortium interest is therefore to combine advantages from both components.

Regarding ethical matrixes (see this page ), the consortium solution presents important advantages:

• It meets the environment well-being values defined by the ethical matrix since the AMP production is accomplished only when triggered by V. cholerea (i.e, it does not create a selection pressure).
• It fulfills the prevention criteria, as water treatment decrease the risk of spreading the pathogen by human vectoring.
• The system is based on the detection of communication molecules meaning that no contact is required between the bacteria target and the effector. This is very important because it validates biosafety requirements of the ethical matrix since the system can be contained in our validated 0.1 μm membrane (see Results ) and still be functional.
• There are no prerequisites to use our technology: no need to add chemical molecules, no need to monitor, no need to gather combustible or electricity to produce energy for thermic or filtration treatment.

3. From wet lab to the field

How we came up with a prototype?

The design of our strategy was carefully designed all along the project. This process started during the initial brainstormings and was enriched by our activities in human practice and integrated human practices. Wetlab and experimental aspects also influenced the project. Our entrepreneurship effort was a determinant contributor to the design and the resulting 3D-printed prototype also imposed some aspect of our technology.

Design evolution evolved mainly with the use and the user. The early design was mainly inspired by our own life experiences and by the SWOT analysis of the project ( described here ). We started with several designs. First, we thought about a cell free solution, interesting to avoid GMM issues. However, it does not profit of the whole advantages of the microbial consortium, as presented above. Second, our partner Sunwaterlife has already developed filtration system to treat water in emerging countries. However, this requires energy. They solved this issue with solar panel, but it means the whole technology to be complex and expensive. We therefore decided otherwise and focuses on a cheap and user-friendly device to contain our engineered microorganisms (described in the ( Device section). Briefly, the device contains our lyophilized strains in a tea-bag-like polymer material allowing diffusion of molecules (including AMP), but not microorganisms. To increase security, this tea-bag is placed in a plastic device with membrane of the same material. It has to be activated in a small volume of water to treat in order to activate the consortium. If the water is contaminated with V. cholerea, the device will release AMPs. Once the active agent is released, the water volume could be increased. The simplicity of the system usage allows easy scale up of the device to meet any volume to treat.

How we defined the end-line customers

There was two antagonists vision when we started the project: the device could be set up for tourist use, with a sport water flask adapted for trekking situation, or the device could be designed to meet a contaminated village requirements. We opted for the second, because our integrated human practice ( see here ) efforts allowed us to define how to provide the solution on the field through NGOs, and because our solution is cheap and easy enough to be used by people with low money or education levels.

How to produce the device?

We were very attentive from the start to the entrepreneurship aspects of the project (see ( see here) ). We felt it would be frustrating to gather and validate science without wondering about the feasibility on the field of our technology. We therefore discussed with industrials, especially from Start-Up companies (( Testimonies here ). They sensibilized us to think at a larger scale for the cycle of life of the product, its sustainability, or the environment in which the product may be used. They also helped us to have a pragmatic approach and explore the market opportunity ((( see here see here).
We have already tested some technical aspect of the project, such has validating the membrane material (( see Results), testing the capacity of V. harvei and P. pastoris to be lyophilized and then co-cultured together, or building a first prototype for the device (( Device section).
Thanks to modeling, we have also demonstrated the feasibility of our strategy under plausible device dimension and microbial concentrations. Our model estimates we have to wait one hour before drinking a non-contaminated water. (see Model) Of course a lot of development works are still required to build a fully functional and reliable device. But with the results we got, we really think it could be a very good investment to pursue the development of our technology. And we are not alone, the SunWaterlife Company showed a high interest in the project and in the development of rapid diagnosis tools to detect cholera.

Conclusion

In a nutshell, our project based on synthetic consortium explores another approach to expand the field of synthetic biology application and remove its limits. The resulting disrupting technology against cholera brings a lot of advantages: conditional activation when needed, good sensitivity, no GMM dissemination, reduction of V. cholerea spreading, cheap usage, low risk of antibiotic resistant strains. The use of synthetic consortium worked well for the cholera thematic, but we feel that such strategies will be more and more common in the iGEM competition and synthetic biology.