Team:INSA-UPS France/test

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Entrepreneurship

Commercialize a novel system for saving lives

With such an innovative system of synthetic communication between microorganisms dedicated to fight cholera, an ongoing human health issue, it would be a nonsense not to exploit it for saving lives.

In this part of the project, our team developed an approach to commercialize our system. To achieve this goal, we first collected testimonies on two topics:

  • a reflexion on the prototype:  how can we best adapt and optimize our system to the current cholera epidemics?
  • an entrepreneurship reflexion: how to set up a company? What shall be our values?

We therefore met successful entrepreneurs in the field of biotechnology and business developer (Marc Lemonnier from the start-up Antabio, Pierre Monsan from Toulouse White Biotechnology, Pierre-Alain Hoffmann from the CRITT Bio-Industries), people that developed a business to treat water, especially from V. cholerae (Christophe Campéri-Ginestet from Sunwaterlife), humanitarians from NGOs that daily face cholera (Claire Salvador from Doctors Without Borders and Alama Keita from UNICEF) and finally, more generally to Westerners. These rich discussions have helped us to deeply understand the cholera context and to adapt our positioning in a viable way for us and sustainable for our targeted users.

Consequently, a scope statement has been established, detailing the functional and technical features of our product.

In parallel, an ethical matrix was built to highlight the important features to work on and to provide a decision tool so that our system is ethically acceptable.

Then, we had to conceive the device following as closely as possible the scope statement: How to best contain GMMs? How to make user-friendly a device? What materials can be used to combine quality and price? Once again, the testimonies and the ethical matrix helped us to reflect about that. Besides, diffusion tests were carried out by our team to choose the best materials of the device. After answering these questions, the device containing our system was modeled and printed in 3D thanks to Jean-Jacques Dumas help, a professional in the design of industrial products.

Finally, once our product created, our team developed a business plan in order to project ourselves into a business creation project, define the action plan to be implemented to exploit this opportunity and how that will result in financial terms. Here, we were assisted by Pierre-Alain Hoffmann, Deputy Director of the business incubator named CRITT Bio-Industries, who has a lot of knowledge in business creation and development.

Entrepreneurship approach to commercialize our project Croc’n Cholera

Testimonies

Scope Statement

Device Conception

Business Plan

  • Scope Statement
  • 1- Project presentation
  • 2- Project requirements
  • Conclusion
  • References

Chapter 1 - Project presentation

Context

Cholera is a diarrhea-causing-disease that has affected the entire world through many epidemics throughout history. Today it still strikes developing countries, war-torn countries or those affected by natural disasters. It is contracted after ingestion of water contaminated with the pathogenic bacterium Vibrio cholerae. The main cause of a cholera epidemic is the lack of drinking water and sanitation resources in affected countries. According to the WHO, between 1.3 and 4 million cases are reported worldwide each year and 21,000 to 143,000 deaths.

Rehydratation is a very efficient treatment to cure the patients, however some people can not get this treatment early enough because they live in remote areas or can not have access to drinkable water. Thus a solution to treat water in these areas has to be found. Current preventive solutions (sterilizing filtration, water treatment by chlorination, etc.) are expensive or difficult to set up.

Purpose of the project

Our team wants to make a new system of detection and purification of water contaminated with the pathogenic bacterium Vibrio cholerae. This device will consist of two elements:

  • A sensor to detect the presence of V. cholerae in water consisting in engineered Vibrio harveyi.
  • An effector to secrete a cocktail of antimicrobial peptides (AMPs) consisting in engineered Pichia pastoris. In Nature, these AMPs are successfully used by the crocodile immune system against bacteria. The peptides used in our system have a broad spectrum of action and are particularly effective against V. cholerae.

The device will be designed for local populations. Thus, the goal is to set up a system as suitable as possible to their users for the purification of water contaminated with cholera.

Organization of the project

This project is run by a group of nine students from different universities: Paul Sabatier University (Toulouse), INSA Lyon and INSA Toulouse. It is supervised by Stéphanie Heux and Brice Enjalbert, researchers at LISBP (Toulouse), assisted by eleven other researchers. The team was formed in January 2017 and will work on this project until November 2017, date of the final restitution at the MIT, Boston.

Chapter 2 - Project requirements

Functional specifications

Detection of Vibrio cholerae

Description

Our system must detect the presence of Vibrio cholerae in water so that the antimicrobial peptides (AMPs) are spread in flow only if it contains Vibrio cholerae. In fact, the production of AMPs has to be controlled for two reasons. On the one hand, if water is drinkable, there is no need for AMPs production. On the other hand, other microorganisms (either from the user microbiota or the nearby environment) won’t develop peptide resistance.

Constraints

To develop the disease, the minimal quantity of V. cholerae cells a human being must ingest is about 104 cells, in one dose of water1, 2. The constraints are the following:

  • to detect this concentration of cells
  • to activate AMP production for water purification only starting from this concentration

Priority

Detection is not the top priority of our system. Purification could work continuously but the implementation of this function is better for safety reason. Moreover, according to the testimonies, the detection function of our system is a new and strong point in the treatment of water contaminated with V. cholerae.

Purification of water

Description

The other aim of our project is to wipe V. cholerae out of water. The system has to produce antimicrobial peptides when water is contaminated with V. cholerae, i.e. when water contains amounts of microorganism superior to the minimum toxic concentration (about 104 cells in one dose of water1). The AMPs used for the project have a broad spectrum of action against microorganisms, but they are particularly efficient against V. cholerae (Leucrocine I : MIC = 0.156 µg/mL3 (>52µg/mL4); D-NY15: MIC = 27 µg/mL4; cOT2: MIC = 29.22 µg/mL5). The goal is to produce these peptides until V. cholerae is wiped out and water becomes drinkable or usable without being dangerous.

Constraints

  • The AMPs have to be correctly synthesized and secreted.
  • They must be stable enough to keep their activities in muddy water for example.
  • The AMPs must properly diffuse to target bacteria.
  • Several factors have to be taken into account: temperature in affected countries (around 40°C (104°F)), the viscosity of water, its pH, etc.

Priority

Purification of water contaminated with V. cholerae is the central part of our project: the priority is high. Without it, which is the first goal of the project, the contract won’t be fulfilled.

Technical specifications

Safety

Ingestion of the GMMs

The exchanges of molecules between the compartment containing GMMs and water must be feasible but the GMMs must not go into water. Actually, on the one hand, the CAI-1 molecule of the V. cholerae quorum sensing must diffuse to the compartment containing the detecting bacteria, V. harveyi. On the other hand, the AMPs produced by P. pastoris must go from the compartment containing the GMMs to the water contaminated with V. cholerae. However, class I GMMs must be kept out of the purified water.

Spreading of the GMMs

GMMs must not be spread in the environment. The compartment containing them must be impact- and break-resistant. People need to be aware that GMMs should not be disseminated in the environment.

Human toxicity of the AMPs

AMPs will be produced and released in water with the aim of wiping out V. cholerae. The amount of AMP remaining in water should not be toxic to humans. Experimentations must be done to know the quantity of AMPs in the water supposed to be drunk and especially their effects on epithelial cells (esophagus, stomach, intestines, etc.).

Limiting media leaks

The proportion of nutrients should be correctly calculated so that they are all consumed during the lifetime of GMMs. An excess of nutrients in the compartment could then diffuse into the water and cause the emergence of new microorganisms.

Environmental

Waste management of the GMMs

As GMMs are used in this system, it is essential to think about their waste management. After the use of the GMMs for the detection of V. cholerae and the purification of water, they must be killed before throwing them away. The questions are: how to kill them and where to dump the GMMs?

Recyclable plastic

In order to have an eco-friendly approach, the plastic used for the device must be recyclable.

Material

GMMs storage

GMMs (V. harveyi and P. pastoris) must be confined in the same compartment because they need to interact with each other. The microorganisms must be kept in this compartment. If they are freeze-dried, the water to be treated must be able to rehydrate the microorganisms. Therefore, water must be able to enter into the compartment.

To sum up, the compartment containing the microorganisms must hold them back but also, allow exchanges of CAI-1 molecules, AMPs and water between the two compartments of the device.

Easy to use

In developing countries, war-torn countries or those affected by natural disasters, people cannot possess a complex device from both a scientific and technical point of view.

Transportable

The device must be easy to transport in order to reach the populations of remote villages or in conditions of natural disaster and armed conflict.

Treatment speed

The treatment speed must be reasonable compared to the volume of treated water.

Water treatment capacity

The water treatment capacity must be adapted to the device function and to the capacity of the microorganisms to detect and treat the water. According to Alama Keita (UNICEF), our device must be able to purify around 11,025 L of water in a week for a village.

Strength

The device has to be built with robust materials (refer to paragraph “Spreading of the GMMs”).

Water taste

Water treatment must not modified its taste so that the product can be easily accepted.

Economical

The price must be adapted to similar options offered on the market.

Chloramine tablets are often used to treat water. In order to be competitive on the market, it is necessary to bring our price into line with our competitors. Therefore, to give us an order of magnitude, cost of chloramine tablets to treat the same volume of water as our product can be calculated.

The product “Chloramine 60 Tablets from SANOFI AVENTIS BELGIUM”6 contains 60 tablets of chloramine and costs 3.69€. According to the instructions, 1 to 4 tablets must be dissolved in 25L of water, depending on the water pollution degree. One tablet costs 3.69/60=0.0615€. Thus, treating 25 L of water costs up to 246cts (4 tablets) and up to 9.84cts for 1L.

To conclude, treating water with our system should not cost more than 10cts per L.

Conclusion about the scope statement

To conclude, the system must be able to detect V. cholerae and release AMPs from a threshold concentration of pathogenic bacterium in water. The water treatment aspect thanks to AMPs is essential.

The device that we have to create must gather technical criteria from an environmental, safety, material and economic point of view that we have listed in the scope statement. These specifications were defined taking into account the advice we received from the people we met throughout our project.

The design of the device will have to best fit these constraints to be accepted by our potential future customers.

References

  1. Baron, S. (Ed.). (1996). Medical Microbiology (4th ed.). Galveston (TX): University of Texas Medical Branch at Galveston.
  2. Nelson et al. (2009). Modeling multiple ecosystem services, biodiversity conservation, commodity production, and tradeoffs at landscape scales. Front Ecol Environ, 7(1): 4-11.
  3. Pata, S., Yaraksa, N., Daduang, S., Temsiripong, Y., Svasti, J., Araki, T., & Thammasirirak, S. (2011). Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Developmental and Comparative Immunology, 35(5), 545–553. https://doi.org/10.1016/j.dci.2010.12.011
  4. Prajanban, B.-O., Jangpromma, N., Araki, T., & Klaynongsruang, S. (2017). Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins. Biochimica Et Biophysica Acta<, 1859(5), 860–869. https://doi.org/10.1016/j.bbamem.2017.01.035
  5. 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. The Journal of Antibiotics, 67(3), 205–212. https://doi.org/10.1038/ja.2013.114
  6. Newpharma. Chloramine 60 Comprimés de SANOFI AVENTIS BELGIUM.
  • Business Plan
  • 1- Status and organization

Business Plan

Now that we have defined in more detail the prototype of our product, we wanted to carry out the entrepreneurship approach by making a complete business plan. A prerequisite to starting a new business is to analyze the market and find the best way to get into it. We would like to warmly thank Mr. Hoffmann, deputy director of CRITT Bio-Industries who helped us in the development of this business plan. He was able to enlighten us on certain points thanks to his expertise in the creation and development of companies in the field of biotechnology processes.

Chapter I - Status and organization

Legal status

Our project would be developed at first as a start-up. Some legal statuses are more adapted to start-ups than others. The french status SARL (Société A Responsabilité Limitée, corresponding to Limited Liability Company) has some drawbacks: the capital is shared in social parts, without any distinctions of profiles. New associates cannot simply join the social parts: complex procedures are needed. The status is not enough flexible for the creation of a start-up.

In contrast, the status of SAS (Société par Actions Simplifiée, which has no english equivalent but could be translated as a “simplified limited liability company”) seems much more flexible and suitable to start-ups. Different social parts associated with different rights are available, the governance body can be modified and finally, there are lots of liberties for status definitions as well as for the arrival of new associates.