Having listed the features our device must display, we started thinking about its design to contain our synthetic biology system. We went over the technical specifications one by one and we tried to find the most suitable solution.
To provide a proof of concept of the feasibility of our product, we first made a prototype. This prototype has been designed to treat 5 liters of water in a single use. It will be necessary in the future to increase the treatment capacity to meet the water needs of cholera-affected villages.
Our synthetic biology system must be confined in a compartment that prevents modified microorganisms from spreading in water and in Nature. So we first thought up a sachet containing our sensor Vibrio harveyi and our killer-effector Pichia Pastoris. This sachet will also contain nutrients to allow microorganism to survive and fulfill their function.
Sachet containing our synthetic biology system composed of V. harveyi and P. pastoris with nutrient medium (Solidworks modeling)
Containment materials
(1) In order to ensure the feasibility of the system and the functional specifications, it is necessary to allow exchanges of CAI-1 molecules and antimicrobial peptides through the sachet. Water must also diffuse in order to rehydrate the freeze-dried microorganisms.
(2) However, GMMs must remain inside the sachet in order to avoid spreading in Nature and ingestion by the users.
(3) To ensure optimum confinement, the chosen material must be strong and must not appear fragile.
(4) Finally, we must find the best cost while also considering the environmental aspect. Materials with a low impact on the environment and recyclable would be ideal.
The solution we chose is a material with pores such as a membrane with an appropriate cut-off threshold.
TPXⓇ, or PolyMethyl Penten (PMP), has already been used in iGEM as a biological system confinement material (Groningen 20121, Toulouse 20152). TPXⓇ consists in thin, transparent, heat-sealable sheets. The volume of the desired sachet is therefore adjustable according to the treatment capacity of the device. It has pores with a diameter of 20 nm. The Toulouse 2015 iGEM team carried out tests to confirm that E. coli BW 25113 grows under TPXⓇ membranes as well as in a culture tube and to attest that bacteria remain inside the sachet after 27 h. V. harveyi has approximately the same size as E. coli (about 2 μm) and P. pastoris cells are about 5 μm in diameter. The following table and figure compare the sizes of the elements.
Estimated diameter of CAI-13 and the AMP4
Element
Estimated diameter (nm)
CAI-1
0.415
Leucrocine I
1.23
D-NY15
15.98
cOT2
19.28
Comparison between the element size: TPXⓇ pores, CAI-1, AMPs and microorganisms
The TPXⓇ pore size is much smaller than microorganisms and larger than CAI-1 and the Leucrocine I AMP. However, they are approximately the same size as the D-NY15 and cOT2 AMPs. This may slow down or avoid their diffusion.
According to the diffusion experiment conducted with TPXⓇ membranes, neither the water nor the Bengale pink dye mimicking the AMPs diffuse through them. Therefore, even if TPXⓇ ensures the containment of our GMMs, we concluded that this material is not suitable for our project.
Another solution came from our meeting with Christophe Campéri-Ginestet, CEO of Sunwaterlife. He suggested to use the membranes that constitute their water filters. These membranes have a cutoff threshold of 0.1 μm which prevents microorganisms from passing through but allows CAI-1, AMPs molecules diffusion (see the following figure).
Comparison between the elements size: Sunwaterlife membrane pores, CAI-1, AMPs and microorganisms
Here, the membrane pore size will not prevent diffusion of D-NY15 and cOT2 AMPs. A diffusion experiment was also performed with these membranes. The dye is able to diffuse through it in less than 1 minute. This means that this material is fully suitable for our project since the 0.1 µm porosity allows peptides diffusion but prevents bacterial transfer. Its permeability towards microorganisms doesn’t need to be tested as they are already used with success in the industry for this purpose. Moreover, it is permeable to water since the start-up company Sunwaterlife uses it for this application. This will allow the perfect rehydratation of the freeze-dried microorganisms. As a results, we will create a sachet containing the freeze-dried microorganisms using this membrane.
→ (1) and (2) are fulfilled.
However, even if we feel confident about the containment properties of the Sunwaterlife membrane, the non-rigid aspect and thinness of the sachet could repel the users. In collaboration with Sunwaterlife, we have designed a solid container for the sachet. It is a cylinder with Sunwaterlife membrane technology as sections. A seal and an external part to be screwed against the membrane will allow its fixing.
Device containing the sachet with Sunwaterlife membrane technology as sections (Solidworks modeling)
The sachet is therefore placed into the cylinder which is then screwed. The whole device can be soaked into the water to be treated. This device ensures a high level of security regarding GMM containment in our system. This gives credibility to our system and it will be easier to get it accepted by clients.
The material initially chosen for the cylinder is ABS (Acrylonitrile Butadiene Styrene) which is a common thermoplastic polymer. It is a light but rigid material, sustainable, water resistant, cheap and shock-resistant. Indeed, it is used to make motorcycle helmets because of its affordability and robustness. Moreover, it can easily be molded, which gave us the opportunity to print it in 3D.
→ (3) is fulfilled.
Finally, ABS is a cheap material: around 1,000€/ton6. It can be used for food applications. Unlike polycarbonate that may contain bisphenol A, a hormone disruptor, ABS is known to be safer and more stable. Therefore, this material can be put in contact with water that will be used by the clients without danger.
The membranes were provided to us free of charge by Sunwaterlife through a bilateral collaboration. They can be put in contact with water safely given that Sunwaterlife uses them to purify water.
In order to limit waste, the sachet must be dimensioned in order to treat as much water as possible in a single use. The cylinder can be reused with a new sachet, reducing the device cost. Sunwaterlife membranes within the cylinder can be changed when they are clogged. Moreover, ABS is a recyclable plastic.
However, these products are not biodegradable. This prospects should be considered in the future.
→ (4) is partially fulfilled.
Device size
The size of the sachet was defined by the modelling approach. A sachet of 20 mL that contains 1.4 g of microorganisms would allow to treat 1 liter of water in approximately 54 minutes (this time does not include the rehydration phase of the microorganisms). As a proof of concept, we decided to create a prototype that contains 100 mL and treats 5 liters of water in approximately the same time (assuming that the water tank is homogeneous at all points).
In order to best satisfy the demand of a village of 15 families and to reduce waste as much as possible, it will be necessary to optimize the treatment capacity in a single use.
The cylinder must be adjusted to the size of the sachet. It should not be too large for convenient use and have approximately the same size as the sachet. However, it should not be too small either because the diffusion rate of water and small molecules (CAI-1 and AMP) across the Sunwaterlife membranes depends on their total surface.
For the prototype, we chose a cylinder which is 54 mm in diameter and 114 mm in length.
3D modeling of our device (Solidworks modeling)
Sachet content
To allow long-term storage of the sachets, the microorganisms and the medium will be freeze-dried. When the device will be immersed in water, the lyophilisate will be rehydrated and the microorganisms will be activated again. However, according to the modeling experiment conducted, the latency phase due to freeze-drying is really long (more than 10 hours for both microorganisms). It will be necessary to optimize this parameter by controlling the amount of microorganisms or their physiological condition before freeze-drying them.
The culture medium should not contain products that are toxic for the environment or humans. According to culture tests, V. harveyi and P. pastoris grow well in LB. This simple medium can be used as a base. However, the complexity of P. pastoris genetics requires a more complex medium, including an ideal glucose concentration of 4% to activate pGAP and a nitrogen deprivation in order to allow the activation of pFUS by Ste12. Glucose concentrations higher than that in LB should not disturb the growth of V. harveyi, but nitrogen deprivation is likely to be a problem. Experiments to optimize the biological system or the medium will be required in a later phase of the project.
We should also make sure that the medium contained in the sachet does not diffuse importantly into the water to change its taste or to allow other microorganisms to develop therein. Once again, a more thorough modeling is necessary.
Operating instructions
One of the biggest challenges for our device to be accepted and widely used is its simplicity. The materials used, robust but light, make it easily transportable.
The cylindrical device is made of 4 parts. The two parts at the extremities allow to hold the Sunwaterlife membranes by screwing them. The seals avoid damaging the membranes and ensure watertightness. The sachet is then placed into the cylinder and the two parts are screwed to contain the sachet. The device can then be soaked into the adequate volume of water to be treated (tank, bucket,...).
At the end of the recommended treatment period, the device must be removed from water that can be used and drunk. The cylinder must be unscrewed and can be reused, as the seals and the membranes if they are undamaged. The sachet can either be thrown into a designated trash bin until the organization comes to treat the waste or burned to destroy the microorganisms. In the first case, NGOs or armies must regularly pick up this bin at the predicted time intervals depending on the processing capacity of the device.
The video below provides a virtual presentation of the handling of our device.
Demonstration of the device use (Solidworks)
To allow a better understanding of the device use, two operating manuals have been made for NGOs and for populations. You can find them here, in the Utilization section. They will be delivered with the device.
3D modeling and printing
As shown in the previous figures, a 3D modeling of the device was carried out on Solidworks when the features were defined. Moreover, Jean-Jacques Dumas, a teacher in industrial products design, helped us in this task, especially with respect to the fixation of the Sunwaterlife membranes, its reusable capacity and its simplicity of use. This allowed us to better imagine and adapt it to the requirements of the customers.
To go further, the device was printed in 3D with a "Stratasys" 3D printer, used for industrial applications. The material used to print the prototype is ABS (acrylonitrile butadiene styrene) which is a plastic polymer that is simple to machine and shock-resistant.
Modeling with Solidworks
Machining parts in the 3D printer
Finally, we are proud to present you our device:
Conclusion about the device conception
The device has been designed to meet as many criteria of the scope statement as possible. Containment of GMMs is achieved using membranes provided by Sunwaterlife. The materials used, the size and the content of the sachet have been studied in order to create a cheap, easy to use and portable device. A prototype of the product was modeled and printed in 3D. With a bag of 10 cL containing 7 g of both microorganisms, it can treat 5 liters of water in about 1 hour (excluding the rehydration time of the microorganisms and assuming that the water is homogeneous).
Obviously, some areas need to be improved:
the use of biodegradable materials;
the treatment capacity that should reach about 11,000 L of water in one week for a village of 18 families. A scale up of this prototype is needed;
if the time required to rehydrate and activate the microorganisms is longer than the diffusion time of the medium’s nutrients, over-dilution will occur, creating an osmotic shock and killing the microorganisms. The rehydration and activation steps must be carried out in a smaller volume of water, set to avoid nutrient over-dilution. Then, once activated, the device can be transferred into a larger volume of water;
in order to optimize the diffusion of water and molecules through the Sunwaterlife membranes, it would be necessary to equip the tanks of water to be treated with stirrers. This would reduce the water treatment time. However, this is difficult to implement in cholera-affected villages and this would make our product too complicated to use.
