Production of tunable nanomaterials using hierarchically assembled bacteriophages

This protocol describes how to design and produce hierarchically assembled nanomaterials with tunable functionalities using engineered M13 bacteriophages. Large-scale fabrication of precisely defined nanostructures with tunable functions is critical to the exploitation of nanoscience and nanotechnology for production of electronic devices, energy generators, biosensors, and bionanomedicines. Although self-assembly processes have been developed to exploit biological molecules for functional materials, the resulting nanostructures and functions are still very limited, and scalable synthesis is far from being realized. Recently, we have established a bacteriophage-based biomimetic process, called 'self-templating assembly'. We used bacteriophage as a nanofiber model system to exploit its liquid crystalline structure for the creation of diverse hierarchically organized structures. We have also demonstrated that genetic modification of functional peptides of bacteriophage results in structures that can be used as soft and hard tissue-regenerating materials, biosensors, and energy-generating materials. Here, we describe a comprehensive protocol to perform genetic engineering of phage, liter-scale amplification, purification, and self-templating assembly, and suggest approaches for characterizing hierarchical phage nanostructures using optical microscopy, atomic-force microscopy (AFM), and scanning electron microscopy (SEM). We also discuss sources of contamination, common mistakes during the fabrication process, and quality-control measures to ensure reproducible material production. The protocol takes ∼8–10 d to complete.