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 the time to find out. However, the first step would then, of course, be to try the ICA method with new streptavidin beads.
As soon as a 4 monomer silk construct is produced, we would insert it into E. coli and purify it 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): 651-663..
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): 341-355.. 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 on this page.
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 of 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 proper 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
PHB should be used as the envelope for our Bacto-Aid. To do this, we first need to optimize the production of PHB even further. This can be done by changing the current gene (phaCAP) in the BioBrick from Tokyo Tech 2012 with a stronger promoter and an additional stronger RBS. The use of a weak promoter and an additional weak RBS might also be an aspect in how much PHB there can be produced in the cell. The new device should then be added to our secretion system instead of the old from Tokyo Tech 2012 BioBrick, and hopefully make the E. coli produce even more PHB than we see now.
To produce more rentable PHB, we should first consider which microorganism to use to make the mass production of PHB most efficient. We have considered using Ralstonia eutropha and Bacillus subtilis, which are two commonly used organisms in the industry. Secondly we need to develop our production so it fits with a continuous stirred-tank reactor. We will imply this reactor for large scale production, so we will be able to regulate parameters such as temperature, oxygen level and amount of required nutrients for the chosen organism. It will likewise also be good if we could decrease the use of chemicals to make the production more profitable.
We also need to design a 3D model of our envelope for Bacto-Aid, so we could print the model and produce the envelope through 3D printing. This should be done by creating 3-5 models of an envelope for Bacto-Aid and the best envelope will be selected critically by important criterias for the use of PHB on the skin and how well it fits these criterias.
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 sub elements up to the industrial scale.