A palette of bright and photostable monomeric fluorescent proteins for bacterial time-lapse imaging

Abstract

Fluorescent proteins (FPs) are pivotal for examining protein production, localization, and dynamics in live bacterial cells. However, the use of FPs in time-lapse imaging is frequently constrained by issues such as oligomerization or limited photostability. Here, we report the engineering of four new fluorescent proteins: mChartreuse (green), mJuniper (cyan), mLemon (yellow), and mLychee (red), using site-directed mutagenesis. These fluorophores outperform current standards for photostability and aggregation properties while retaining high levels of brightness in living E. coli bacteria. Starting with superfolder GFP (sfGFP), we developed mChartreuse, which shows significantly enhanced brightness, stability, and monomericity compared to other GFP variants. From mChartreuse, we derived mJuniper, a cyan fluorescent protein with rapid maturation and high photostability, and mLemon, a yellow fluorescent protein with improved photostability and brightness. We also identified a mutation that eliminates residual oligomerization in red fluorescent proteins derived from Discosoma species, such as mCherry and mApple. Incorporating this mutation into mApple, along with other substitutions, resulted in mLychee, a bright and photostable monomeric red fluorescent protein. These novel fluorescent proteins advance fluorescence time-lapse analysis in bacteria and their spectral properties match current imaging standards, ensuring seamless integration into existing research workflows.