Team:INSA-UPS France/Applied Design

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Applied design


Overview
A real world problem
A microbial consortium
From wet lab to the field
Lifecycle & impact
Conclusion
References

Overview: the way from synthetic biology to real life

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 consortium to detect and eliminate Vibrio cholerae in drinkable water. We modified three different organisms in the course of this project and the results are very promising. We managed to establish synthetic communication between Escherichia coli and Vibrio harveyi. We also successfully engineered the yeast Pichia pastoris to produce efficient antimicrobial peptides from crocodile! Modeling also provided promising informations regarding the global behavior of our consortium.

Fig. 1: the way from synthetic biology to real life: an integrated engineering procedure.

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 taking into account pieces of advice 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 beginning point of this project since the Sunwaterlife Company has already mentioned its interest to develop our system.

The design of our strategy evolved all along the project. This process started during the initial brainstormings and was enriched by our activities in human practice and integrated human practices, such as testimonies and discussions with citizens. Wetlab, experimental and modeling aspects also influenced the project. Our entrepreneurship effort was a determinant contributor to the design and the resulting 3D-printed prototype also impact some aspect of our technology.

1 - Solve a real world problem: cholera, a widespread disease…

Gathering knowledge about cholera

Cholera is a worldwide diarrheal disease, caused by the ingestion of V. cholerae 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.

Fig. 2: countries reporting cholera deaths and imported cases in 20161

The WHO reported over 1 million cases over the year 2015 and the mortality was around 1%2. 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 population3 in the long-term. This fact highlights 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.

Fig. 3: problem tree of the cholera disease (template from Synenergene, the iGEMer’s Guide to the Future4).

A problem tree is very useful to have a global representation of the cholera background. The causes are located on the roots and the effect are in the foliage. The goal is to identify the root-cause and to solve it using synthetic biology. Many of the causes are related to water: insufficient water supply, unsafe drinking water for instance. This is quite logical as water is one of Vibrio cholerae life medium. If the water access is not safe, cholera continue to spread. What parameters conditionate the water quality? Water treatment and the external contamination like fluids coming from contaminated people. Indeed the human can be a true bioreactor for V. cholerae growth and multiplication. Other causes are also related to water like natural catastrophes and war which make water treatment facilities unusable.

All together these causes demonstrate that water treatment can be a true solution to contain cholera epidemia. This accession is confirmed by Webber et al. 20055 and Bardhan, n.d.6, expecting that the improvement of the water treatment quality can possibly reduce the cholera occurrence by 90% whereas the improvement of sanitation will have a moderate impact. The NGO Doctors Without Borders, in their guidelines, argue that the increase of purified water disponibility greatly reduces the attack rate (the ratio between reported cases on the number of peoples exposed to cholera).

Working on water treatment is to us the most pertinent way to contribute to the fight against cholera.

Analyzing the current solutions

Therapeutics

Fig. 4: currently used therapeutical solutions against cholera.

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 symptoms7. 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. cholerae reservoir and can contribute to spread the disease. Moreover, all humans resulting fluids have to be considered as Biosafety 2 contaminated wastes.

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 efficiency8. Efficient scientific detection or purification methods exist, but they require trained staff and sophisticated equipment which are generally not available in all areas of the world8.

Water treatment

Fig. 5: currently used methods for water purification.

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 be 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.9 Besides, bleach impacts the organoleptic properties of the water, hence presents an acceptability problem. However, bleach is current best solution available so far, according to the WHO (2014)

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 reserve is contaminated again because of people use. Thus, new methods of prevention and treatment have to be developed.

This is why our system has been designed to treat water in most cases. 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 implement in the all-day life, and has a minimal impact on organoleptic characteristic of the water.

Fig. 6: iGEM teams which have already worked on cholera: St Andrew 2010, Dundee Schools 2016, Colombia 2014 and Sheffield 2010.

We were not the first iGEM team to engage in the cholera fight. The main challenge of previous iGEMers was about V. cholerae quorum sensing detection. They worked with E. coli as a chassis but all faced major issues to detect V. cholerae quorum sensing since transposition of signaling pathway between microorganisms is a complex task, even harder if we want to integrate a bacterial pathway in a yeast. That was our main reason to choose the cholera problem 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 an innovative molecule is a great asset to the project. This is reinforced by the expression conditional to V. cholerae presence. D-NY15 use will also be facilitated by the fact that it does not show any cytotoxicity effect on human cells.10

Advantages of the consortium solution

Fig. 7: our multi-organisms system to fight against cholera.

Building a synthetic microbial consortium with V. harveyi 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. cholerae 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.

Table 1: decisions-taking process for a microbial consortium.
Decisions Well-being Autonomy Justice
Environment well-being Biosafety requirements Prevention criteria
No prerequisite to use our technology

Regarding ethical matrixes (see Integrated Human Practices), 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 prerequisite 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

Fig. 8: evolution of our prototype design through our engineering process. Enlarge

How we came up with a prototype?

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 by V. cholerae, 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.

Fig 9: our 3D printed protection cylinder.

How we defined the end-line customers

There was two antagonist visions 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. Moreover, the imported cholera cases into developed countries are rare.11 We can assess that the methods deployed to protect the tourists in endemic areas are enough efficient in comparison with the number of local people contaminated by the cholera.

How to think and produce the device? Working with industrials.

Fig. 10: meeting with the Deputy Director of CRITT Bio-Industries (Regional Center for Innovation and Technological Transfert in the field of Bio-Industries).

We were very attentive from the beginning to the entrepreneurship aspects of the project (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 startup (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).

We have already tested some technical aspect of the project, such has validating the membrane material (see Results), testing the capacity of V. harveyi 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. However, 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.

4 - How its lifecycle can more broadly impact our lives and environment?

As our system contains synthetic microorganisms, containment has to be the priority during the life cycle to protect the human and the environmental integrity. Two main aspects can be studied: the waste treatment and the surveillance of the permeability of the prototype outer membrane. Those features are critical to ensure the safety of the user.

A systemic approach was achieved to investigate and evaluate the impact of our technology on the social, economical and environmental field at every step of the life cycle. Our disrupting system introduces new practices both for local people and NGOs with a more rigorous waste treatment strategy. Indeed, synthetic organisms are present in the device and has to be properly treated adding other steps at the end of the life cycle. The NGOs have to master the use of the system before deploying it on the field and then to engage in end-user training process.

The GMMs discharging risk is still present but is greatly limited by the use of a double protection. The impact on the daily life as to be as softer as possible. Nevertheless if the project shows convincible results, the acceptability will probably greatly increase. As a result, it will impact conception of the synthetic biology which will be assimilated to a robust technology.

To learn more see our human practice gold page.

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 GMMs dissemination, reduction of V. cholerae 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.

References

  1. WHO. Countries reporting cholera deaths and imported cases in 2016. http://www.who.int/gho/epidemic_diseases/cholera/cases/en/
  2. Ali M, Nelson AR, Lopez AL & Sack DA (2015) Updated Global Burden of Cholera in Endemic Countries. PLOS Neglected Tropical Diseases https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4455997/
  3. WHO. Yemen crisis. (27 september 2017 update) http://www.who.int/hac/crises/yem/en/
  4. SYNERGENE, RATHENEAU INSTITUT, The iGEMers guide to the future. 2016. https://live.flatland.agency/12290417/rathenau-igem/
  5. Webber, R. (2005). Communicable disease epidemiology and control – A Global Perspective. 2nd edition. Wallingford: CABI Publishing.
    6. http://www.harep.org/Agriculture/2005p.pdf
  6. Bardhan, P.K. (n.d.). Cholera Outbreaks: Impact, Prevention, Control. International Centre for Diarrhoeal Disease Research, Bangladesh
    7. http://www.paho.org/disasters/index.php?option=com_docman&task=doc_download&gid=1706&Itemid=1179.
  7. Davies HG, Bowman C & Luby SP (2017) Cholera - management and prevention. Journal of Infection. 74 Suppl 1:S66-S73 http://www.journalofinfection.com/article/S0163-4453(17)30194-9/pdf
  8. Clemens JD, Nair GB, Ahmed T, Qadri F & Holmgren J (2017) Cholera. The Lancet http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(17)30559-7/abstract
  9. Castle Chemicals, Sodium Hypochlorite Stability, Technical data bulletin, http://castlechem.com.au/sds/Sodium-Hypochlorite-Stability.pdf
  10. Yaraksa N, Anunthawan T, Theansungnoen T, Daduang S, Araki T, Dhiravisit A & Thammasirirak S (2014) Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts. Journal of Antibiotics 67 205 https://www.ncbi.nlm.nih.gov/pubmed/24192554
  11. Morger, H., Steffen, R. and Schar, M. (1983). Epidemiology of cholera in travellers, and conclusions for vaccination recommendations. Br Med J, 286(6360), pp.184-186.
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