Date of Award
Doctor of Philosophy (PhD)
Production of novel protein-based materials has become a widespread and valuable field of research within synthetic biology. There are many protein-based materials found in nature, such as dragline spider silk, squid sucker ring teeth, and dragonfly resilin, that are attractive due to their extraordinary material properties, which include high tensile strength and elasticity. Some creatures in nature produce protein materials that allow them to exhibit the fascinating phenomenon of adhesion. Geckos use setae, or microhairs, on their toes to stick to and climb up walls, while chameleons and slugs secrete sticky, viscous mucus that trap prey or predators. Remarkably, many marine organisms, such as barnacles and sandcastle worms exhibit adhesion, but underwater. These creatures can adhere to surfaces to protect themselves from predators and intertidal forces. Mussels have a unique mechanism for exhibiting strong underwater adhesion. Mussels secrete adhesive threads, or byssi, that contain proteins known as mussel foot proteins (Mfp). These proteins have inspired the development of adhesives, such as Cell-TakTM that can adsorb to wet surfaces that commercial adhesives, such as cyanoacrylates, polyurethanes, and epoxies, cannot. However, these wet adhesives are produced in low yields, are extremely expensive, and are limited in application. Thus far, there are too few, if any, viable bulk adhesives that can be produced at a large scale and adhere strongly enough to be used in underwater repair and biomedical applications. Recombinant engineering and production is a sustainable and economically feasible method that can allow for the synthesis of tunable bulk underwater adhesives with desirable mechanical properties and in high yields.
The first aim of this dissertation is to develop a new strategy in synthetic biology that can produce high molecular weight oligomers of Mfp5, the most adhesive Mfp subtype in the mussel byssus, in part due to its high 3,4-dihydroxyphenylalanine content. The developed technology, which uses split inteins to splice smaller Mfp subunits together, allows for the production of Mfp of higher molecular weights, a feat that had previously been impossible to achieve at the transcriptional & translational levels alone. By producing these protein adhesives, a positive correlation between molecular weight and underwater adhesion strength was observed, which leads to a better understanding of the biophysical and biochemical mechanisms that underlie underwater adhesion of Mfp5, but also the Mfp network within the mussel byssus as a whole.
Next, the Mfp5 domain was explored further for its potential in engineering applications using two approaches. Mfp5 was used as a polymeric matrix for graphene oxide nanofiller for the production of high-strength and ultra-tough composite materials that are comparable to or exceed the strengths of similar graphene oxide-based materials, as well as many metals, ceramics, and polymers. Secondly, the Mfp5 domain was fused to structural motifs from amyloid and spidroin proteins in order to synthesize free-standing bulk underwater adhesives that can easily be delivered to and cure between a variety of surfaces underwater. The results presented in this dissertation highlight the power of synthetic biology tools for the heterologous expression of mussel-inspired polymeric materials that can be used in the synthesis of composites and bulk underwater adhesives for a variety of applications.
Janie Brennan, Marcus Foston, Srikanth Singamaneni, Yinjie Tang,
Available for download on Tuesday, August 13, 2030