Engineering and characterization of new LOV-based fluorescent proteins from Chlamydomonas reinhardtii and Vaucheria frigida.

Flavin-based fluorescent proteins (FbFPs) are a new class of fluorescent reporters that exhibit oxygen-independent fluorescence, which is a key advantage over the green fluorescent protein. Broad application of FbFPs, however, has been generally hindered by low brightness. To maximize the utility of FbFPs, there is a pressing need to expand and diversify the limited FbFP library through the inclusion of bright and robust variants. In this work, we use genome mining to identify and engineer two new FbFPs (CreiLOV and VafLOV) from Chlamydomonas reinhardtii and Vaucheria frigida. We show that CreiLOV is a thermostable, photostable, and fast-maturing monomeric reporter that outperforms existing FbFPs in brightness and operational pH range. Furthermore, we show that CreiLOV can be used to monitor dynamic gene expression in Escherichia coli. Overall, our work introduces CreiLOV as a robust addition to the FbFP repertoire and highlights genome mining as a powerful approach to engineer improved FbFPs.

Automated single cell microbioreactor for monitoring intracellular dynamics and cell growth in free solution.

We report an automated microfluidic-based platform for single cell analysis that allows for cell culture in free solution with the ability to control the cell growth environment. Using this approach, cells are confined by the sole action of gentle fluid flow, thereby enabling non-perturbative analysis of cell growth away from solid boundaries. In addition, the single cell microbioreactor allows for precise and time-dependent control over cell culture media, with the combined ability to observe the dynamics of non-adherent cells over long time scales. As a proof-of-principle demonstration, we used the platform to observe dynamic cell growth, gene expression, and intracellular diffusion of repressor proteins while precisely tuning the cell growth environment. Overall, this microfluidic approach enables the direct observation of cellular dynamics with exquisite control over environmental conditions, which will be useful for quantifying the behaviour of single cells in well-defined media.

Zooming in on biological processes with fluorescence nanoscopy.

Fluorescence nanoscopy enables the study of biological phenomena at nanometer scale spatial resolution. Recent biological studies using fluorescence nanoscopy have showcased the ability of these techniques to directly observe protein organization, subcellular molecular interactions, structural dynamics, electrical signaling, and diffusion of cytosolic proteins at unprecedented spatial resolution. Super-resolution imaging techniques critically rely on bright fluorescent probes such as organic dyes or fluorescent proteins. Recently, these methods have been extended to live cells and multicolor, three-dimensional imaging, thereby providing exquisite spatiotemporal resolutions of the order of 10-20 nm and 1-2 s for subcellular imaging. Further improvements in image processing algorithms, labeling techniques, correlative microscopy, and development of advanced fluorescent probes will be required to achieve true molecular-scale resolution using these techniques.