Project Summary Materials for applications in healthcare and medicine usually come from two main groups (a) synthetic polymers specifically designed to achieve a certain goal or (b) naturally derived biopolymers that are leveraged in their native or slightly modified state for a specific goal. The advantage of being a synthetic chemist is that technically, if you can synthesize it, the possibilities are infinite, but the downfall is that often solvents or portions of the polymer cause cytocompability issues or concerns when it comes to translation and implantation in a human. Alternatively, unmodified natural biopolymers, or proteins, have an easier path toward Food and Drug Administration's approval, but lack the customizability afforded in synthesis or chemical modification. Genetic engineering via production of small peptides in bacteria has improved the availability of customizable short peptides, but proteins on the order of hundreds of kilodaltons cannot be produced this way. This is the case for the silk fibroin biopolymers isolated from caterpillars in the Lepidoptera order, where the heavy chain of silk fibroin is known to be over 300 kilodaltons in length. Genetic engineering, using tools such as CRISPR or PiggyBac, provides an avenue for theoretically modifying the sequence of silk proteins, which has been attempted with limited success in the domesticated silkworm, Bombyx mori. However, silk is collected from the cocoon of the B. mori pupae, meaning that the life cycle of this silkworm is interrupted, making it difficult to maintain these modified populations or assess phenotypes in a high-throughput manner. To address this, silk fibroin will be isolated from an entirely different silk-producing species: Plodia interpunctella, or the Indianmeal moth. Under specific conditions, this agricultural pest produces sheets of silk prior to entering the cocooning phase. These easily collectable sheets of silk fibers can then be cleaned, degummed, and regenerated to an aqueous biopolymer solution. Moreover, unlike B. mori, P. interpunctella silk collection does not interrupt the life cycle of the silkworm/moth and these silkworms are easier to stably genetically modify though embryo injections compared to B. mori. In this Maximizing Investigators' Research Award, genetic engineering will be leveraged to modify the silk fibroin protein sequence at the organismal level, adding in new peptide sequences such as mammalian cell binding motifs or sites for human growth factor sequestration. Scale-up of the process will be achieved via transcriptional regulation of silk fibroin as a function of external stimuli such as humidity or pathogens. Together, these two strategies for enhancing the bio-functionality of the silk fibroin protein and the scale-up required for advanced manufacturing of medical devices or materials will be explored. The outcomes of this work include full biophysical, biochemical, and in vivo characterization of these materials throu...