An easy method for quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins

Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays. However, they introduce significant inaccuracies in studies involving microaerobiosis or anaerobiosis, as oxygen is required for the maturation of these proteins' chromophores. In this study, the authors highlight the errors incurred by using fluorescent proteins under limited oxygenation by comparing standard fluorescence-based reporter gene assays to quantitative real-time PCR data in the study of a complex oxygen-regulated gene network. Furthermore, a solution to perform quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins using a microplate reader with an oxygen control system and applying pulses of full oxygenation before fluorescence measurements is provided. Strains expressing fluorescent proteins to be compared in a reporter gene assay are grown in 96-well plates and incubated in a microplate reader with oxygen-control capabilities. Rather than determining fluorescence under microaerobic or anaerobic conditions, strains are first grown to the desired optical density under full oxygenation, followed by a long incubation under the oxygenation condition of choice and fluorescence is measured after a final pulse of 21% O2 to allow for chromophore maturation.

Automated summary

Fluorescent proteins introduce inaccuracies in studies involving limited oxygenation, but a solution using a microplate reader with oxygen control system is provided to quantify gene expression under anaerobic and microaerobic conditions.