A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.
A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.
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DOI：
10.1038/s41589-018-0004-9
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发表时间：
2018-04
影响因子：
14.8
通讯作者：
Boyden ES
中科院分区：
文献类型：
--
作者：
Piatkevich KD;Jung EE;Straub C;Linghu C;Park D;Suk HJ;Hochbaum DR;Goodwin D;Pnevmatikakis E;Pak N;Kawashima T;Yang CT;Rhoades JL;Shemesh O;Asano S;Yoon YG;Freifeld L;Saulnier JL;Riegler C;Engert F;Hughes T;Drobizhev M;Szabo B;Ahrens MB;Flavell SW;Sabatini BL;Boyden ES
在这里，我们提出了一种新的方法来工程复杂的蛋白质向多维规格，通过一个简单而可扩展的定向进化策略。通过机器人挑选在显微镜下被鉴定为表达同时表现出几种特定特性的蛋白质的哺乳动物细胞，我们可以在几个小时内筛选文库中的数十万种蛋白质，沿着沿着多个性能轴评估每一种蛋白质。我们通过识别一种新的遗传编码的荧光电压指示剂，同时使用我们的显微镜引导的细胞挑选策略优化蛋白质的亮度和膜定位来展示这种方法的强大功能。我们生产了高性能的基于视蛋白的荧光电压报告Archon 1，并通过在急性小鼠脑切片以及在体内的幼斑马鱼中成像尖峰和毫伏级亚阈值和突触活动来证明其实用性。我们还展示了在C.优雅的。
We here present a new way to engineer complex proteins toward multidimensional specifications, through a simple yet scalable directed evolution strategy. By robotically picking mammalian cells that are identified, under a microscope, to express proteins that simultaneously exhibit several specific properties, we can screen through hundreds of thousands of proteins in a library in a matter of a few hours, evaluating each along multiple performance axes. We demonstrate the power of this approach by identifying a novel genetically encoded fluorescent voltage indicator, simultaneously optimizing brightness and membrane localization of the protein using our microscopy-guided cell picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1, and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices as well as in larval zebrafish in vivo. We also demonstrate measurement of postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.
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DOI：
10.1039/c4ib00251b
发表时间：
2015-02
期刊：
Integrative biology : quantitative biosciences from nano to macro
影响因子：
--
作者：
Dean KM;Lubbeck JL;Davis LM;Regmi CK;Chapagain PP;Gerstman BS;Jimenez R;Palmer AE
通讯作者：
Palmer AE
影响因子：
16.6
作者：
Gong, Yiyang;Wagner, Mark J.;Li, Jin Zhong;Schnitzer, Mark J.
通讯作者：
Schnitzer, Mark J.
影响因子：
64.8
作者：
Grigoryan, Gevorg;Reinke, Aaron W.;Keating, Amy E.
通讯作者：
Keating, Amy E.
DOI：
10.1126/science.aab0810
发表时间：
2015-12-11
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gong Y;Huang C;Li JZ;Grewe BF;Zhang Y;Eismann S;Schnitzer MJ
通讯作者：
Schnitzer MJ
液滴微流体流式细胞仪，用于对遗传编码传感器的瞬时细胞反应进行分类。
影响因子：
7.4
作者：
Fiedler BL;Van Buskirk S;Carter KP;Qin Y;Carpenter MC;Palmer AE;Jimenez R
通讯作者：
Jimenez R