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 Results ) and still be functional.

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.