Directed Evolution of a Genetically Encoded Bioluminescent Ca 2+ Sensor

Engineering Proceedings

article

Yufeng Zhao¹  Sungmoo Lee¹  Robert E. Campbell²  Michael Z. Lin¹ 

[1] Department of Neurobiology, Stanford University;Department of Chemistry, The University of Tokyo;Department of Chemistry, University of Alberta;Department of Bioengineering, Stanford University;Department of Chemical and Systems Biology, Stanford University

关键词: Ca2+ sensor;    directed evolution;    bioluminescence;   

DOI  :  10.3390/IECB2023-14563

来源: mdpi

【 摘 要 】

The use of genetically encoded fluorescent sensors for the calcium ion (Ca2+) has revolutionized neuroscience research by allowing for the recording of dozens of neurons at the single-cell level in living animals. However, fluorescence imaging has some limitations such as the need for excitation light, which can result in a highly auto-fluorescent background and phototoxicity. In contrast, bioluminescent sensors using luciferase do not require excitation light, making them ideal for non-invasive deep tissue imaging in mammals. Our lab has previously developed a bioluminescent Ca2+ sensor CaMBI to image Ca2+ activity in the mouse liver, but its responsiveness to Ca2+ changes was suboptimal. To improve the performance of this sensor, we applied directed evolution to screen for genetic variants with increased responsiveness. Through several rounds of evolution, we identified variants with more than five times improved responsiveness in vitro. We characterized the improved sensors in culture cell lines and dissociated rat neurons and confirmed that they exhibited a higher sensitivity to changes in intracellular Ca2+ levels compared to their progenitor. These optimized Ca2+ sensors have the potential for non-invasive imaging of Ca2+ activity in vivo, particularly in the brain.

【 授权许可】

Unknown