2020 Fiscal Year Annual Research Report
Directed Evolution of a Palette of Optogenetic and Chemi-Optogenetic Indicators for Multiplexed Imaging of Cellular Metabolism
| Project/Area Number |
19H05633
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| Research Institution | The University of Tokyo |
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Principal Investigator |
キャンベル ロバート.アール 東京大学, 大学院理学系研究科(理学部), 教授 (40831318)
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| Co-Investigator(Kenkyū-buntansha) |
合田 圭介 東京大学, 大学院理学系研究科(理学部), 教授 (70518696)
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| Project Period (FY) |
2019-06-26 – 2024-03-31
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| Keywords | Protein Engineering / Fluorescence / Microscopy / Cell Biology |
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| Outline of Annual Research Achievements |
In FY2020 our efforts have been concentrated on two parallel approaches: 1) Fully optogenetic biosensors; and 2) Hybrid chemi-optogenetic biosensors. In the area of fully optogenetic biosensors Assistant Professor Yusuke Nasu and B4 student Saaya Hario developed red and green biosensors of intracellular and extracellular lactate with performance that far exceeds that of any previously reported lactate biosensor. In addition, Master’s student Rina Hashizume created two new near-infrared Ca2+ biosensors. In the area of chemi-optogenetic biosensors, Master’s student Hayato Kadoya successfully created a PYP (photoactive yellow protein)-based biosensor for Ca2+. Furthermore, Doctoral student Wenchao Zhu has developed a new design of HaloTag based biosensor with specificity for Ca2+. We are particularly excited about the prospects for this novel design which is based on a synthetic ion chelator that is used to label a HaloTag-GFP hybrid protein. New Master’s student Dazhou Cheng, who joined in September 2020, is making excellent progress on yet another distinct HaloTag-based biosensor design, and is specifically focussed on the development of chemi-optogenetic biosensors for Ca2+ and K+.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Overall, we making very good progress towards achieving the major goals of our KIBAN(S) grant. One of our initial major goals was to develop the extracellular lactate sensor, and this has now been achieved. We are now just waiting on biological results from our international collaborators. Another major achievement has been the development of proof of concept of chemi-optogenetic biosensors. When we started this project, it was not clear how effective such a novel and innovative design would be. With our success at making both HaloTag-based and PYP-based biosensors, we have now demonstrated that this technology has tremendous potential. Some other aspects of the project are proceeding a bit slower than expected. In particular, our efforts to develop chemi-optogenetic NIR indicators are going a bit slower than planned. This has largely been due to our focus on first focussing our efforts on chemistry-optogenetic biosensor designs. Now that such designs have been established in our lab, we will begin to shift our focus towards adapting them to work with NIR fluorophores.
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| Strategy for Future Research Activity |
For FY2021, the lab will be highly focussed on further development of all of these distinct and unique biosensor designs. For the extracellular lactate biosensors, we are now far advanced in exploring novel biological applications and expect to publish a high profile manuscript in FY2021. For the intracellular lactate biosensors, we are only just starting the biological applications and so this will likely remain our focus through all of FY2021. For the NIR Ca2+ optogenetic Ca2+ biosensors, we will begin exploring biological applications of the one known as iBB-GECO, while the variant known as dNIR-GECO will still require further engineering through FY2021. For chemi-optogenetic biosensors, Zhu’s project is now at an advanced stage and we expect to be wrapping it up and submitting a manuscript in FY2021. A B4 student, Takeuchi has joined the lab from April 1, 2021 and will further explore Zhu’s novel biosensor design. Specifically, they will attempt to apply it for development of Na+ sensors. All other chemo-genetic biosensor projects in the lab are at a more preliminary stage and we will stay focused on technological development and improvement through FY2021.
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[Presentation] A cellular morphological indicator toward directed evolution of microalgae for highly efficient wastewater treatment2020
Author(s)
M. Xu, D. Yuan, S. Yan, C. Lei, K. Hiramatsu, J. Harmon, Y. Zhou, M. H. Loo, T. Hasunuma, A. Isozaki, and K. Goda
Organizer
化学とマイクロ・ナノシステム学会
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[Presentation] 出芽酵母の大規模な遺伝子解析のための高スループット細胞形態分取法2020
Author(s)
H. Matsumura, A. Isozaki, Y. Zhao, K. Huang, T. Wakamiya, D. Yuan, H. Mikami, S. Ohnuki, S. Uemura, Y. Ohya, and K. Goda
Organizer
化学とマイクロ・ナノシステム学会
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