Prospects

How would we make Bacto-Aid?

Even though we spent hundreds of hours in the lab, we did not end up standing with the Bacto-Aid in our hands in Boston. But do not worry - we got a plan for how you can end up with our bandage. On this page we will enlighten you about the next steps in our project, which we would have followed if the days contained more than 24 hours or if we had a bunch of effective minions available. These steps have to be fulfilled before we can create a full functional patch that can be used in hospitals and in the everyday life.

Making a 4 monomer silk construct

There were some difficulties with producing a longer silk construct and whether it was due to the reproducibility of UCLA’s method or the too old streptavidin beads, we did not have time to conclude on. However, the first step would then of course be to try the ICA method with new streptavidin beads. When we have produced a 4 monomer silk construct, we would be inserting it into E. coli. and purify our silk from the bacteria. Here we would have liked to try a purification method without the use of His-tag, because earlier literature have demonstrated that recombinant silk produced without a His-tag have better mechanical properties compared to the fibres made from silk proteins with a His-tag Tokareva, O., et al. (2013). "Recombinant DNA production of spider silk proteins." Microbial biotechnology 6(6): 651-63.. For the spinning of silk we would first have tried to spin our silk by wet spinning, which is a very simple and inexpensive method Teulé, F., et al. (2013). "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." Nature protocols 4(3): 341-55.. In relation to using the recombinant silk for wound healing, it could also be interesting to prepare a three dimensional scaffold of the silk. Here a number of methods, such as salt leaching, gas forming or freeze-drying, have been reported to generate porous three-dimensional matrices Nazarov, R.., et al. (2004). "Porous 3-D Scaffolds from Regenerated Silk Fibroin" Biomacromolecules 5: 718-727.. The detailed description of how we want to proceed with a 4 monomer silk construct can be reviewed here.

The hybrid silk fibre

The idea of the hybrid silk fibre was to incorporate bacteriocins between silk monomers. From the literature it is known that silk does not lose its function when other proteins are combined with the silk Gomes, S. C., et al. (2011). "Antimicrobial functionalized genetically engineered spider silk." Biomaterials 32(18): 4255-4266.. We also found that proteins incorporated in silk monomers does not lose their functions either Gomes, S. C., et al. (2012). "Biological responses to spider silk-antibiotic fusion protein." Journal of tissue engineering and regenerative medicine 6(5): 356-68.. This is why we believe that if the bacteriocins were to be incorporated in the silk monomers, then both components would maintain their functions and thereby their effects. We are also interested in creating hybrid silk with more than one bacteriocin incorporated to see if they have a better effect than single bacteriocins between the silk. Our hypothesis is that silk fiber, which is proven to be immune neutral and promotes wound healing, combined with the antimicrobial effect from the bacteriocins, would create a synergistic effect resulting in decreasing infections and also a decrease in healing time in patients with severe wounds. We tried to ligate the bacteriocin together with a silk fragment, but we did not succeed despite having the correct overhangs. The next step will then be to make more experiments with the goal of making a successful ligation between the bacteriocins and a silk construct. In theory we should succeed, so it might just take some more trials before we do.

Improving the production of PHB

Producing Bacto-Aid

If we have fully developed our sub elements, we should be able to make Bacto-Aid and test its effect on wound healing. The next step would then be to develop methods for how we could scale our production of the subelements up to the industrial scale.