Directed Evolution of a Palette of Optogenetic and Chemi-Optogenetic Indicators for Multiplexed Imaging of Cellular Metabolism

在2022财年，我们报告了许多高性能的基因编码和化学遗传学生物传感器，这些传感器在过去几年里一直在我们实验室开发中。例如，改进的绿色和红色遗传编码生物传感器，针对细胞内和细胞外乳酸成像进行了优化，在BioRxiv上发布的一系列三个预印本中报告了这些生物传感器(2022.12.27.522013；2022.10.19.512892；和2022.08.30.505811)。为了证明我们的新型生物传感器的实用性和重要性，我们与生物学家合作，使用它们来研究乳酸在内质网-线粒体相互作用中的作用(在细胞报告中描述)。在另一项工作中，我们在《蛋白质科学》杂志上描述了一种改进的基因编码的远红荧光钙离子生物传感器。这些出版物包括几个合作和创新的例子，将新的生物传感器应用于多路复用成像。在化学遗传指示剂方面，我们在《自然化学生物学》上报道了我们的基于人工合成的钙、钠离子螯合剂和绿色荧光蛋白的化学遗传指示剂。除了已发表的工作外，我们还在继续探索和进一步优化这一新设计。在2022财年，坎贝尔举办了19场关于KIBAN(S)研究的特邀研讨会，包括在SPIE光子学西部的主旨演讲，在生物成像学会年会上的主旨演讲，在巴黎芬斯论坛的会议演讲，以及在牛津大学的系级演讲。坎贝尔实验室的学员们还做了许多关于KIBAN(S)项目的海报和口头演示。

In FY2022 we reported many of the high performance genetically encoded and chemigenetic biosensors that had been under development in our lab over the past few years. For example, improved green and red genetically encoded biosensors, optimized for imaging of intra- and extracellular lactate, were reported in a series of three preprints posted to bioRxiv (2022.12.27.522013; 2022.10.19.512892; and 2022.08.30.505811). To demonstrate the utility and importance of our new biosensors, we collaborated with biologists who used them to investigate the role of lactate in endoplasmic reticulum-mitochondria interactions (described in Cell Reports). In other work we described an improved genetically-encoded far-red fluorescent Ca2+ biosensor in Protein Science. This publications included several collaborative and innovative examples of applying the new biosensor to multiplexed imaging. In the area of chemigenetic indicators, we reported our chemigenetic indicators based on synthetic Ca2+ and Na+ chelators and green fluorescent protein in Nature Chemical Biology. Beyond the published work, we are continuing to explore and further optimize this new design. During FY2022, Campbell gave 19 invited seminars on KIBAN(S) research, including a keynote at SPIE Photonics West, a keynote at the Annual meeting of the Bioimaging society, a conference presentation at the FENS Forum in Paris, and a Departmental presentation at Oxford University. Trainees in the Campbell lab gave many additional poster and oral presentations on KIBAN(S) projects.