This project was chosen due to the interest in synthetic spider silk. An article of interest concluded that synthetic spider silk fused to human defensins was stable while preserving the antimicrobial effect of the defensins, thus it became one of the founding pillars of our project Gomes, Silvia C, Isabel B Leonor, Joao F Mano, Rui L Reis og David L Kaplan (2011). “Antimicrobial functionalized genetically engineered spider silk.” eng. I: Bio- materials 32.18, s. 4255–4266. issn: 1878- 5905<. The idea of making PHB a part of our project as the plastic in the bandage, evolved from the desire to solve the growing problem of plastic pollution. Bacto-Aid was the perfect project to make our two wishes come to life.
The three major subparts of our project are thoroughly described in the following sections.
The bacteriocins are the antimicrobial peptides in our hybrid silk. Resistance development has been creating difficulties in treating infections with traditional antibiotics. Since no resistance has been observed against bacteriocins that have been used the past 50 years, they show great potential as an alternative treatment. The bacteriocins act bactericidally through pore formation and interfere with intracellular enzymatic reactions of the specific targeted bacteria Zendo, T., Yoneyama, F., & Sonomoto, K. (2010). Lactococcal membrane-permeabilizing antimicrobial peptides.<. Bacteriocins are proteins, but are not used today for therapeutical use due the denaturation in the stomach. Intravenous administration has been tested in vivo and shows promising results Lohans, C. T., & Vederas, J. C. (2012). Development of Class IIa Bacteriocins as Therapeutic Agents. International Journal of Microbiology, 2012, 13. doi:10.1155/2012/386410<. As both bacteriocins and silk are proteins, we can incorporate bacteriocins into the silk polymer through gene-manipulation. We, and the iGEM team of Stockholm, performed MIC-tests to quantify the effect of our bacteriocins against resistant pathogenic bacteria. The tests show that purified bacteriocins were quite effective as growth inhibitors. For further information about bacteriocins, read here.
Spider silk was chosen due to its angiogenic properties and proliferative effect on keratinocytes (skin cells), which is not seen in the traditional used gauze Fredriksson, C., Hedhammar, M., Feinstein, R., Nordling, K., Kratz, G., Johansson, J., . . . Rising, A. (2009). Tissue Response to Subcutaneously Implanted Recombinant Spider Silk: An in Vivo Study. Materials, 2(4), 1908.<. The silk is produced natively by Nephila clavipes via repetitive coding sequences of the major silk components, MaSp1 and MaSp2 (Major Spidroin 1 and 2). In order to obtain the ability to create silk, these repetitive regions have been introduced into the E. coli via a plasmid. Synthesizing repetitive sequences are troublesome, and is difficult to do through the iGEM standard 3A assembly, due to multiple scar-sites. To avoid scar-sites we tried to synthesize the silk with the protocols provided by the 2015 iGEM team from UCLA, because they succeeded in producing spider silk from bacteria. The method allows the creation of a hybrid silk consisting of bacteriocins and spider silk. For further information about the spider silk, read here.
Poly-β-hydroxybutyrate (PHB) is a biosynthesized polyester, belonging to a group of biodegradable plastics called polyalkanoates (PHA). PHB is non-toxic and has a high oxygen-permeability JAMBUNATHAN, P. & ZHANG, K. C. 2016. Engineered biosynthesis of biodegradable polymers. Journal of Industrial Microbiology & Biotechnology, 43, 1037-1058.<. The combination of different promoters and ribosomal binding sites have previously been shown to positively affect the yield of PHB. The yield has further been increased by the introduction of the pantothenate kinase BioBrick. By introducing a PHB secretion system into an E. coli strain, we can possibly achieve a continuous production of PHB from the existing plastic producing BioBricks. Our system has already left us with enough PHB to 3D print a part of a human jaw. The secretion system we introduce, use phasin as a transportermolecule for the secretion of PHB. Phasin binds to PHAs, which creates the possibility of its use when working with other types of biodegradable PHA polyesters in bacteria Hanisch, J., et al. (2006). "The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins." Microbiology 152(Pt 11): 3271-3280.. For further information about PHB, read here.
In the field of science, scientific reproduction is one of the most important aspects when confirming a hypothesis or theory. iGEM follows this specific scientific virtue by making it a part of both the bronze and gold criteria: the demands imply working with other team’s work. We took it further trying to reproduce parts of earlier iGEM team’s work: the 2015 UCLA team, the 2012 Tokyo Tech team, the 2013 Imperial College London and the 2015 Standford Brown team.