Hernandez, Eric2022-09-132022-09-132022-06https://hdl.handle.net/11299/241559University of Minnesota M.S. thesis. June 2022. Major: Microbial Engineering. Advisor: Kyle Costa. 1 computer file (PDF); vi, 49 pages.Live-cell fluorescence imaging in methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) when complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F420-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate containing medium, consistent with previous studies suggesting increased abundance of these proteins in the absence of H2. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed membrane localization in single cells observed using anoxic fluorescence microscopy. Additionally, split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Lastly, we demonstrate an example in which FAST is able to validate the localization of an uncharacterized protein and provide a framework for future multi-color imaging that would empower further studies going forward. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. Portions of this thesis have been submitted for publication in The Journal of Bacteriology.enFASTFluorescenceMethanogenThesis or Dissertation