Scope statement

The scope statement is divided in two main chapters: the project presentation and the project requirements. In that entrepreneurship part, our goals are:

• to define clearly the context, the purpose and the organization of the project;
• to describe all the functional and technical specifications that our product must meet.

The testimonies and reflection made through the ethical matrix were taken into account to define the requirements of the product. Once all that points mentioned, the reflection about the design of the product can begin.

Chapter 1 - Project presentation

I. 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 cholera epidemics 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, causing 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 solutions including sterilizing filtration, treatment by chlorination, etc. are expensive or difficult to set up.

II. Purpose of the project

Our team wants to make a new system to detect the pathogenic bacterium Vibrio cholerae in water and purify the contaminated water to make it drinkable. This device will consist of two elements:

• A sensor to detect the presence of V. cholerae in water consisting in an engineered Vibrio harveyi strain;
• An effector to secrete antimicrobial peptides (AMPs) consisting in an engineered Pichia pastoris strain. 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 user-friendly as possible for the purification of water contaminated with V. cholerae.

III. Organization of the project

This project is run by a group of nine students from three 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

I. Functional specifications

1. Detection of Vibrio cholerae

A. Description

Our system must detect the presence of Vibrio cholerae in water so that the antimicrobial peptides (AMPs) are produced and released 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’s microbiota or the nearby environment) won’t develop peptide resistance.

B. 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 water^{1, 2}. The constraints are the following:

• to detect this concentration of cells;
• to activate AMPs production for water purification only starting from this concentration.

C. 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 novel and important point in the treatment of water contaminated with V. cholerae.

2. Purification of water

A. 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 microorganisms 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 so that water becomes drinkable or usable without danger.

B. 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.

C. 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 reached.

II. Technical specifications

1. Safety

A. Ingestion of the GMMs

The exchanges of molecules between the contaminated water and the compartment containing GMMs and producing the AMPs must be efficient but the GMMs must not go into water. For the detection system, the CAI-1 molecule of the V. cholerae quorum sensing mechanism must diffuse from contaminated water to the compartment containing the detecting bacteria V. harveyi. For the purification system the AMPs produced by P. pastoris must diffuse from the compartment containing the GMMs to the contaminated water. However, class I GMMs must be kept out of the purified water.

B. Spreading of the GMMs

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

C. Human toxicity of the AMPs

AMPs will be produced and released in water with the aim of wiping out V. cholerae. The amount of AMPs 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.) and in the microbiota.

D. Limiting media leaks

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

2. Environmental

A. Waste management of the GMMs

As GMMs are used in this system, it is essential to think about waste management. After the use, the GMMs must be inactivated before discarding them as regular garbage. The important questions to consider are: how to kill them and where to discard GMMs?

B. Recyclable plastic

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

3. Material

A. GMM 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 rehydrate the microorganisms. Therefore, water must enter into the compartment.

B. Easy to use

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

C. 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 conflicts.

D. Treatment speed

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

E. 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 water. According to Alama Keita (UNICEF), our device must allow to purify around 11,025 liters of water in a week for a village.

F. Strength

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

G. Water taste

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

4. Economical

The retail price of our device 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 in line with our competitors. Therefore, to give us a general idea, the cost of chloramine tablets to treat the same volume of water as our device 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 24.6cts (4 tablets) and up to 0.91cts for 1 L.

To conclude, treating water with our system should not cost more than 1cts per L.

Note: our system use a more advanced technology than chloramine tablets. It allows not only to treat but also to detect the presence of V. cholerae. It appears justifiable to take into account a higher water treatment price with our product.

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.