While the packaging of the product is only the final aspect to think of for other projects, it is a pivotal element for us. Of course, the product needs to meet biosafety requirements, but our device also has to fulfil special challenges for our vision of the S.H.I.E.L.D.

The final version of the S.H.I.E.L.D. will consist of the upper part including cyanobacteria. Since they are dependent on sunlight the upper part needs to be transparent. The bottom part contains our modified E. coli. These two components are linked by a connective part (Fig.1). The device needs to be designed to protect both - the GMOs and the surrounding environment. All parts are described in the following.

Fig. 1 S.H.I.E.L.D.

As our device is employing a spatially divided co-culture, it was important to meet the demands of both. For one thing, we needed to assure optimal conditions for the cyanobacteria S. elongatus, which we were provided to us by the iGEM Team Düsseldorf within the framework of our collaboration. These bacteria are functioning as nutrient suppliers for the E. coli, which are in turn responsible to lure and kill malaria mosquitoes. As cyanobacteria are in need of sunlight as an energy source to be able to produce glucose, they are located in the upper part of the S.H.I.E.L.D. To ensure sufficient access to sunlight, the cyanobacteria are surrounded by a transparent acrylic glass dome. The cells are separated and immobilised by alginate capsules, which allow for diffusion of glucose and avoid sinking and accumulation of the bacteria on the ground of the funnel. Through a vertical connector, the produced nutrient is transferred to the E. coli, which are located at the bottom. To maintain an even distribution of the E. coli culture, we took advantage of iGEM Eindhoven’s provided solution: a novel 3D gel-embedded culture. We employ their dextran-based gel as matrix for E. coli. Thus, the bacteria are immobilised by the expression of matrix adhering membrane proteins and sedimentation is avoided. The dextran gel is remarkably stable for an extended amount of time under varying conditions, which exceed tolerance of most common bacteria. Moreover, the gel has additional inhibiting effects on the cells, complementing our soft growth inhibition module, ensuring maximum sustainability. Using this culture technique, we are confident in sustainability far beyond classic culture technologies could provide in the field.

Apart from arrangements for an optimal co-culture, biosafety is of great relevance. For this purpose, we integrated a commercially available nanofilter separating both the cyanobacteria compartment from the E. coli compartment underneath, and the contained GMO from the environment. While preventing microorganisms from both exiting or entering the trap, the filters pore size does allow for the diffusion of lures and toxin. On the other hand, the filter keeps the S.H.I.E.L.D. free of of incoming pathogens through mosquito stings into the gels. The diffused odour bait molecules and insecticides that passed the nanofilter are then collected in a poly ammonium salt (PAS) hydrogel, acting as a reservoir. From the surface of the gel, the bait molecules evaporate evenly and lure mosquitoes which can land on the gel’s surface and take up the insecticide by stinging into the gel. With its self-healing properties, the gel remains its integrity even after being stung into. However, if too much water evaporates from the S.H.I.E.L.D., the gel acts as a protective layer to prevent the bacteria culture from drying out. The gel returns to its original characteristics when new water is taken up. To support fast water supply, the device is designed to enable condensation water to be conducted to the hydrogel. An intact hydrogel is not only important for the S.H.I.E.L.D.’s functionality but it also creates a further layer of protection between the GMOs and and the environment.As another important feature, the designed hydrogel is antimicrobial. This is especially important as a fungal film would have several adverse effects. The growth of funghis would inhibit the diffusion of the substances resulting in discouraging the mosquitoes from stinging into the gel. Furthermore, the antimicrobial nature of the hydrogel ensures that pathogens can not be distributed by mosquitoes landing on the gel. Dr. Jacobs, mosquito expert from the Bernhard Nocht Institute for Tropical Medicine, assured us the growth of pathogens on our gel is highly unlikely. Additionally, the hydrogel also guarantees that only animals that can sting the hydrogel and suck the toxin out are impacted by the S.H.I.E.L.D. The specificity of our mosquito trap is further enhanced by the lures used. Further information can be found in the section Safety

Additionally to our GMO tailored safety measures, we used plastic that is very hard, durable, UV resistant and autoclavable in addition to strong sealing rings to form the outer material. Thus we increase the sustainability and simultaneously facilitate the handling.  

To produce the S.H.I.E.L.D.’s first prototype, we applied 3D printing. We are aware the S.H.I.E.L.D.’s final version cannot be produced cheaply and will cost more per piece than other malaria prevention methods, due to the elaborate assembling of the different components. However it will be more profitable considering the time period the S.H.I.E.L.D. will be active. Nevertheless, we designed simple shapes that can easily be assembled and produced in a straightforward way, like injection moulding.