Application of the Fluorescence-Activating and Absorption-Shifting Tag (FAST) for Flow Cytometry in Methanogenic Archaea

Abstract

Methane-producing archaea play an essential role in the global carbon cycle and demonstrate great potential for various biotechnological applications, e.g., biofuel production, carbon dioxide capture, and electrochemical systems. Oxygen sensitivity and high autofluorescence hinder the use of common fluorescent proteins for studying methanogens.

Teaching old dogs new tricks: genetic engineering methanogens

“…More recently, fluorescence-activating and absorption-shifting tags (FAST and FAST2) have been developed that function under anaerobic conditions and have been applied to methanogens, which expands the available molecular tools to include fluorescent-based protein localization, high-throughput flow cytometry, and fluorescence-activated cell sorting ( 57 – 59 ). FAST and FAST2 are fluorogen-activating proteins that cause small fluorogens to fluoresce brightly upon binding with the protein.…”

“…This type of reporter system only requires a small monomeric protein (e.g, 14 kDa, 125 amino acid residues), and can be used with several fluorogens that have different absorption and emission wavelengths ( 60 , 61 ). For example, a FAST2 tag was used in M. acetivorans and M. maripaludis to conduct flow cytometry, which may then be used to quantify target methanogens in multi-population systems ( 57 ). Thus, FAST reporters are a promising tool for accelerating methanogen gene and protein studies.…”