A single-phase flow microfluidic cell sorter for multiparameter screening to assist the directed evolution of Ca2+ sensors
A single-phase flow microfluidic cell sorter for multiparameter screening to assist the directed evolution of Ca2+ sensors
复制标题
用于多参数筛选的单相流微流体细胞分选仪，以协助 Ca2 传感器的定向进化
DOI：
10.1039/c9lc00779b
复制
发表时间：
2019
期刊：
影响因子：
6.1
通讯作者：
Harrison D. Jed
中科院分区：
文献类型：
--
作者：
Zhao Yufeng;Zhang Wei;Zhao Yongxin;Campbell Robert E.;Harrison D. Jed
我们介绍了一种单相流微流控细胞分选器，具有两点检测系统，能够进行双参数筛选，以协助细菌细胞中表达的基于荧光蛋白的Ca2+传感器的定向进化。新的细胞分选系统利用两个荧光显微镜获得信号在两个不同的点沿着流动路径，其中分析物的浓度变化，Ca2+，被诱导。因此，两个探测器确定了传感器在反应后荧光变化的幅度，以及传感器的整体亮度。设计了三维聚焦流，增强了细胞的空间控制和信号对匹配。细胞分选器以中等吞吐量筛选传感器，每秒10个细胞和每轮105个细胞，为后续的人工筛选提供了更高的准确性。与之前基于亮度筛选的单点检测相比，我们的新μFACS大大加速了遗传编码Ca2+传感器的定向进化。两轮定向进化产生了一种名为Y-GECO2f的变体，与进化前的变体相比，其亮度增加了26%，体外Ca2+依赖性荧光变化增加了300%以上。
We introduce a single-phase flow microfluidic cell sorter with a two-point detection system capable of two-parameter screening to assist with directed evolution of a fluorescent protein based Ca2+ sensor expressed in bacterial cells. The new cell sorting system utilizes two fluorescence microscopes to obtain signals at two different points along a flow path in which a change in concentration of the analyte, Ca2+, is induced. The two detectors thus determine the magnitude of fluorescence change of the sensor following the reaction, along with the overall brightness of the sensor. A design for a 3D focusing flow was configured to enhance the spatial control of cells and signal pair-matching. The cell sorter screens the sensors at a moderate throughput, 10 cells per s and 105 cells per round, enriching top variants for the subsequent manual screening with higher accuracy. Our new μFACS greatly accelerates the directed evolution of genetically encoded Ca2+ sensors compared to the previous version with single point detection for brightness-based screening. Two rounds of directed evolution led to a variant, named Y-GECO2f, which exhibits a 26% increase in brightness and a greater than 300% larger Ca2+-dependent fluorescence change in vitro relative to the variant before evolution.