Lustro: High-Throughput Optogenetic Experiments Enabled by Automation and a Yeast Optogenetic Toolkit
Lustro: High-Throughput Optogenetic Experiments Enabled by Automation and a Yeast Optogenetic Toolkit
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DOI：
10.1021/acssynbio.3c00215
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发表时间：
2023-07-11
影响因子：
4.7
通讯作者：
McClean, Megan N. N.
中科院分区：
文献类型：
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作者：
Harmer, Zachary P. P.;McClean, Megan N. N.
光遗传系统使用遗传编码的光敏蛋白来控制细胞过程。这提供了用光正交控制细胞的潜力;然而，这些系统需要许多设计-构建-测试周期来实现功能设计，并且需要费力地调整多个照明变量以获得最佳刺激。我们将联合收割机实验室自动化和模块化克隆方案相结合，以实现酿酒酵母中光遗传学分裂转录因子的高通量构建和表征。我们扩展了酵母光遗传学工具包，包括隐花色素和增强型磁铁的变体，将这些光敏二聚体纳入分裂转录因子，并在96孔微孔板格式中自动照明和测量培养物，以实现高通量表征。我们利用这种方法来合理设计和测试优化增强型Magnet转录因子，提高光敏基因的表达。这种方法可推广到一系列生物系统和应用中的光遗传学系统的高通量表征。
Optogenetic systems use genetically encoded light-sensitiveproteinsto control cellular processes. This provides the potential to orthogonallycontrol cells with light; however, these systems require many design-build-testcycles to achieve a functional design and multiple illumination variablesneed to be laboriously tuned for optimal stimulation. We combine laboratoryautomation and a modular cloning scheme to enable high-throughputconstruction and characterization of optogenetic split transcriptionfactors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants ofthe cryptochromes and enhanced Magnets, incorporate these light-sensitivedimerizers into split transcription factors, and automate illuminationand measurement of cultures in a 96-well microplate format for high-throughputcharacterization. We use this approach to rationally design and testan optimized enhanced Magnet transcription factor with improved light-sensitivegene expression. This approach is generalizable to the high-throughputcharacterization of optogenetic systems across a range of biologicalsystems and applications.