Dual-expression system for blue fluorescent protein optimization.

Area of Science:

• Biotechnology
• Molecular Biology
• Microscopy

Background:

• Spectrally diverse fluorescent proteins (FPs) are crucial for multiplexed biological imaging.
• Existing blue fluorescent proteins (BFPs) often exhibit lower brightness and lack comprehensive characterization for mammalian cell and model organism imaging.

Purpose of the Study:

• To introduce and characterize novel blue fluorescent proteins, Electra1 and Electra2.
• To evaluate the performance of these new BFPs in cultured mammalian cells and in vivo model organisms.

Main Methods:

• Hierarchical screening of new BFPs using a novel dual-expression vector in bacterial and mammalian cells.
• Systematic benchmarking of Electra variants against state-of-the-art BFPs in cultured mammalian cells.
• Validation of Electra variants for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice using one-photon and two-photon microscopy.

Main Results:

• Introduction of Electra1 and Electra2, novel blue fluorescent proteins with enhanced properties.
• Demonstrated utility of Electra variants as fluorescent tags for structural proteins in mammalian cells.
• Successful validation of Electra variants for multicolor in vivo imaging across multiple model organisms, comparable to leading BFPs like mTagBFP2.

Conclusions:

• Electra1 and Electra2 are suitable for multicolor imaging in cultured cells and various model organisms.
• The novel dual-expression vector shows potential for directed molecular evolution of fluorescent proteins.