We believe that the dual-expression vector and the presented method of hierarchical screening can be readily deployed for protein engineering of other novel FPs.ĭevelopment of optimized BFPs using a hybrid expression vector We also demonstrated the utility of Electra1 and Eectra2 for multicolor neuroimaging in vivo in model organisms such as Caenorhabditis elegans, zebrafish, and mice in combination with conventional green and red FPs using one-photon and two-photon microscopy. Electra1 and Electra2 exhibited high performance as fluorescent tags for structural proteins in mammalian cells. To optimize intracellular brightness, we introduced a novel protein optimization approach using a hybrid expression system, which enabled a high-throughput assessment of intracellular brightness in bacterial and mammalian cells. To address these limitations, we engineered two monomeric blue FPs (BFPs), named Electra1 and Electra2, with optimized intracellular brightness and conducted their benchmarking against the top-performing BFPs reported to date. Furthermore, available blue FPs were not systematically characterized in culture mammalian cells and model organisms in vivo. However, blue FPs 20, 21, 25 exhibit significantly lower brightness compared to green, yellow, and red FPs. Currently available monomeric green 17 and yellow 18 FPs are characterized by the highest molecular brightness and are usually selected in combination with red 19 and blue 20, 21 FPs a combination that provides crosstalk-free spectral multiplexing conditions using standard one-photon 8, 22 and two-photon 23, 24 imaging setups. In addition to intracellular brightness, the monomeric state of FPs is considered a prerequisite for many applications, mainly because monomeric FPs are advantageous over their dimeric or tetrameric counterparts since they allow for artifact-free protein-of-interest tagging. , in order to evaluate their performance and provide valuable insight for end-users. This introduces a need for testing newly developed FPs under various experimental conditions including expression vectors, cell types, model organisms etc. However, there is mounting evidence supported by numerous studies that high molecular brightness does not always correspond to high intracellular brightness in cultured cells and in vivo 13, 14, 15, 16. According to FPbase ( ) 12 there are multiple available FPs with high molecular brightness, mostly emitting cyan, green, yellow, and red fluorescence. One of the major criteria when selecting a genetically encoded FP for most in vivo applications is fluorescence brightness, among others 11. For example, investigation of clonal expansion and tissue plasticity as well as neuronal tracing are heavily reliant on spectral diversity of multicolor strategies 9, 10. Spectral diversity of fluorescent proteins (FPs) provides a straightforward approach for multiplex imaging of different subcellular and cellular structures in cultured cells 1, 2, 3, 4 and various model organisms 5, 6, 7, 8. We believe that the described dual-expression vector has a great potential to be adopted by protein engineers for directed molecular evolution of FPs.ĭuring the past few decades, there is an increasing need for simultaneous high-content imaging of multiple subcellular and cellular structures in intact biological systems. The developed BFPs are suitable for multicolor imaging of cultured cells and model organisms in vivo. The Electras variants were validated for multicolor neuroimaging in Caenorhabditis elegans, zebrafish larvae, and mice in comparison with one of the best in the class BFP mTagBFP2 using one-photon and two-photon microscopy. We performed systematic benchmarking of Electras against state-of-art BFPs in cultured mammalian cells and demonstrated their utility as fluorescent tags for structural proteins. Here we introduce a pair of new BFPs, named Electra1 and Electra2, developed through hierarchical screening in bacterial and mammalian cells using a novel dual-expression vector. Furthermore, available BFPs were not systematically characterized for imaging in cultured mammalian cells and common model organisms. Among FPs fitting standard color channels, blue FPs (BFPs) are characterized by lower brightness compared to other spectral counterparts. Spectrally diverse fluorescent proteins (FPs) provide straightforward means for multiplexed imaging of biological systems.
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