Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor
The use of genetically encoded fluorescent sensors for the calcium ion (Ca<sup>2+</sup>) 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...
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MDPI AG
2023-05-01
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| Series: | Engineering Proceedings |
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| Online Access: | https://www.mdpi.com/2673-4591/35/1/20 |
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| author | Yufeng Zhao Sungmoo Lee Robert E. Campbell Michael Z. Lin |
| author_facet | Yufeng Zhao Sungmoo Lee Robert E. Campbell Michael Z. Lin |
| author_sort | Yufeng Zhao |
| collection | DOAJ |
| description | The use of genetically encoded fluorescent sensors for the calcium ion (Ca<sup>2+</sup>) 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 Ca<sup>2+</sup> sensor CaMBI to image Ca<sup>2+</sup> activity in the mouse liver, but its responsiveness to Ca<sup>2+</sup> 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 Ca<sup>2+</sup> levels compared to their progenitor. These optimized Ca<sup>2+</sup> sensors have the potential for non-invasive imaging of Ca<sup>2+</sup> activity in vivo, particularly in the brain. |
| first_indexed | 2024-03-10T22:48:38Z |
| format | Article |
| id | doaj.art-c8b56f92bf214bb1809502ffa21d654c |
| institution | Directory Open Access Journal |
| issn | 2673-4591 |
| language | English |
| last_indexed | 2024-03-10T22:48:38Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Engineering Proceedings |
| spelling | doaj.art-c8b56f92bf214bb1809502ffa21d654c2023-11-19T10:32:09ZengMDPI AGEngineering Proceedings2673-45912023-05-013512010.3390/IECB2023-14563Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> SensorYufeng Zhao0Sungmoo Lee1Robert E. Campbell2Michael Z. Lin3Department of Neurobiology, Stanford University, Stanford, CA 94305, USADepartment of Neurobiology, Stanford University, Stanford, CA 94305, USADepartment of Chemistry, The University of Tokyo, Tokyo 113-0033, JapanDepartment of Neurobiology, Stanford University, Stanford, CA 94305, USAThe use of genetically encoded fluorescent sensors for the calcium ion (Ca<sup>2+</sup>) 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 Ca<sup>2+</sup> sensor CaMBI to image Ca<sup>2+</sup> activity in the mouse liver, but its responsiveness to Ca<sup>2+</sup> 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 Ca<sup>2+</sup> levels compared to their progenitor. These optimized Ca<sup>2+</sup> sensors have the potential for non-invasive imaging of Ca<sup>2+</sup> activity in vivo, particularly in the brain.https://www.mdpi.com/2673-4591/35/1/20Ca<sup>2+</sup> sensordirected evolutionbioluminescence |
| spellingShingle | Yufeng Zhao Sungmoo Lee Robert E. Campbell Michael Z. Lin Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor Engineering Proceedings Ca<sup>2+</sup> sensor directed evolution bioluminescence |
| title | Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor |
| title_full | Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor |
| title_fullStr | Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor |
| title_full_unstemmed | Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor |
| title_short | Directed Evolution of a Genetically Encoded Bioluminescent Ca<sup>2+</sup> Sensor |
| title_sort | directed evolution of a genetically encoded bioluminescent ca sup 2 sup sensor |
| topic | Ca<sup>2+</sup> sensor directed evolution bioluminescence |
| url | https://www.mdpi.com/2673-4591/35/1/20 |
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