A fundamental problem faced in deep biosphere research is the low rates at which metabolic activity is occurring in the subsurface. Protein-targeted studies have only rarely been the center of attention, most importantly due to the lack of methodology to visualize protein synthesis within cells and the need for radioactively or stable isotopically labeled substrates to study bulk protein turnover. In response to these limitations, I have recently developed a novel click chemistry-based approach that uses a bioorthogonal non-canonical amino acid to fluorescently track protein synthesis within cells. Within the proposed project I will further develop this method with the goal to establish protocols for the separation and characterization of translationally active cells as well as for the enrichment and identification of newly synthesized proteins. After establishing these techniques using representative pure and co-cultures, I will refine them using enrichments from Hydrate Ridge methane seep sediment. Furthermore, I will study the applicability of other, yet uncharacterized bioorthogonal amino acids to seep sediments as well as >1,700 meters-below-seafloor deep sandstone samples obtained during IODP-expedition 337. This project directly addresses C-DEBI research themes 1, 3, and 4 and will support an early career researcher who has not previously been funded by C-DEBI.