The California market squid has specialized skin that is able to manipulate light through reversible condensation and assembly of reflectin proteins, allowing the squid to tune its skin iridescence for precise camouflage and underwater communication. Differential phosphorylation of reflectin proteins is observed to be contaminant with the reversible condensation and hierarchical assembly of reflectin proteins. Our mechanistic understanding of the role of protein phosphorylation in reflectin-mediated iridescence remains incomplete due to the inability to obtain large quantities (>lmg) of a homogenous population of phosphorylated reflectins. To overcome this limitation, we propose to leverage state-of-the-art biotechnologies. These include 1) an engineered f. coli strain with an expanded genetic code and 2) an orthogonal translation system capable of site-specific incorporation of phosphoserine into proteins. We will aim to mimic the squid by incorporating phosphoserine into reflectin in a genetically encoded manner. To accomplish this, the learning-objectives-by-doing in this interdisciplinary project will be: cloning the reflectin Al gene, constructing mutants, protein expression in a bacterial system, and purification of site-specifically phosphorylated variants of reflectins. Our work will provide the foundation for elucidating the role of phosphorylation in reflectin-mediated camouflage in marine organisms.
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