Longwei Liu¹, Praopim Limsakul¹, Shaoying (Kathy) Lu¹, Peter Yingxiao Wang^1,*

Molecular & Cellular Biomechanics, Vol.16, Suppl.2, pp. 82-82, 2019, DOI:10.32604/mcb.2019.07360

Abstract Genetically-encoded biosensors based on Fluorescence Resonance Energy Transfer (FRET biosensors) have been widely used to dynamically track the activity of Protein Tyrosine Kinases (PTKs) in living cells because of their sensitive ratiometric fluorescence readout, high spatiotemporal resolution. However, the limitation in sensitivity, specificity, and dynamic range of these biosensors have hindered their broader applications, and there was a lack of efficient ways to optimize FRET biosensors. Here we established a rapid, systematic and universal approach for FRET biosensor optimization through directed evolution which involves generating genetic diversity and screening for protein variants with desired properties at the same time and… More >

Yingxiao Wang^1,*

Molecular & Cellular Biomechanics, Vol.16, Suppl.2, pp. 79-79, 2019, DOI:10.32604/mcb.2019.07268

Abstract Genetically-encoded biosensors based on fluorescence proteins (FPs) and fluorescence resonance energy transfer (FRET) have enabled the specific targeting and visualization of signaling events in live cells with high spatiotemporal resolutions. Single-molecule FRET biosensors have been successfully developed to monitor the activity of variety of signaling molecules, including tyrosine/serine/threonine kinases. We have a developed a general high-throughput screening (HTS) method based on directed evolution to develop sensitive and specific FRET biosensors. We have first applied a yeast library and screened for a mutated binding domain for phosphorylated peptide sequence. When this mutated binding domain and the peptide sequence are connected by… More >