Development of Functional Materials Based on Engineered Filamentous Bacteriophage via Site-Specific Modification and 3D Printing Technology

Abstract

Filamentous bacteriophage (notably f1, fd, and M13) have demonstrated exciting properties as functional materials or scaffolds in biomedical and nanomaterial technology applications. Because of their structures they have been implemented as flexible nanofiber structures and have also been employed as liquid-crystalline biomaterials with capabilities including molecular recognition, catalysis, self-assembly, and nanoparticle nucleation. In addition to chemical modification of the filamentous bacteriophage to alter their properties, the coat proteins of the phage can be easily modified by genetic encoding to control the chemical nature and function of the phage, since they are produced by growth and release from Escherichia coli (E. coli). Moreover, because filamentous bacteriophage are produced by infective growth from E. coli, the phage can be made simply and in economical large quantities. In this study, new strategies were proposed for the development of functional materials and scaffolds through the site-specific modification of coat proteins on filamentous bacteriophage, immobilization of materials onto the phage as well as immobilization of the phage itself onto patterned substrates, and fabrication of various forms of 3D structures using the phage as the lone structural material. The general modification strategy used in this work for virus engineering proves to be of great interest for labeling the phage with a range of amine or carboxylic acid functionalized chemical targets. Even more exciting is the use of site-specific modification which can be used to incorporate unnatural components into the phage display screening process which has been used as a biotech discovery tool. Aside from site-specific modification, this work also utilized native bacteriophage to show that metal ion precursors could be effectively used to form a self-assembled phage nanowire network. This work proved that native phage p8 binds to cobalt cations and the resulting nanowire network was catalytically active. Furthermore, in this work the ability to form not only self-assembled nanowires but also customized patterned nano/microfibers by a 3D meniscus guided printing approach provided a significant improvement to our understanding of the mechanisms of generating liquid-crystalline 3D viral micro/nano architectures. The possibility of generating free-form architectures from filamentous bacteriophage was demonstrated in sensor and actuator device structures as well as in patterned structures for culture and growth of living mammalian cells. These foundational results using purely biological structural components may play an important role for future sensor development for harmful environmental vapors or even possibly as scaffolds for biomimetic tissue engineering. Through modification of the filamentous phage with azobenzene derivatives, it was further confirmed that stimuli responsive photo-switching materials could be successfully constructed. Covalent labeling of phage with the azobenzene derivatives under optimal conditions where the phage structure could be stably preserved allowed the resulting filamentous phage material (termed Azo-M13) to adopt alternative confirmations in response to UV irradiation. As such, this phage material exhibited reversible deflection of the azo-m13 structure by UV light and visible light and may serve as a photo-actuating material to be implemented for systems in which morphological changes by light response are desired. This work has proven unique concept in biomaterials fabrication, which may be useful for investigators researching topics especially on virus coat protein. Furthermore, this highly effective technique for virus engineering may thus be easily customized for a number of applications by allowing selective bio conjugation of a wide range of chemical targets. Beyond the molecular level, 3D printed fabrication of nano/microscale scaffolds and device architectures from these modifiable phage opens the way for new development using engineered bacteriophage. In future, this new approach offers the necessary innovative link with nature's adaptive systems in order to generate future micro fabricated functional materials.