A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting
A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting
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基于荧光激活细胞分选的甲基萘醌精确定量高通量筛选策略
DOI：
10.1007/s10295-016-1757-3
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发表时间：
2016-06-01
影响因子：
3.4
通讯作者：
Zheng, Zhi-ming
中科院分区：
文献类型：
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作者：
Liu, Yan;Xue, Zheng-lian;Zheng, Zhi-ming
为了提高对一株高产菜籽酮(维生素K-2，MK)菌种的筛选效率和准确性，建立了一种新的荧光激活细胞分选(FACS)定量方法。染色技术被优化，以最大限度地扩大自发细胞和MK积聚细胞之间的荧光信号差异。荧光载体罗丹明123(Rh123)具有反映膜电位的能力，是一种将MK含量与荧光信号定量连接的合适的荧光染料。为了促进荧光分子与靶分子的充分接触并保持较高的细胞存活率，对染色和孵育条件进行了优化。结果表明，10%的蔗糖促进了Rh123的摄取，同时保持了一定的细胞活力。在Rh123染色前用氯化镁预处理细胞也能提高细胞存活率。利用流式细胞仪，在不到1h的时间内可以很容易地检测到5万个细胞，优化的染色方法可以得到平均荧光与高效液相色谱测定的MK含量的线性响应。我们开发了一种新的、有用的染色方法来高通量评价黄杆菌。突变体库，使用流式细胞仪来鉴定具有增加的MK积累特性的突变体。本研究也为其他工业微生物菌株的筛选提供了参考。
To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K-2, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl2 before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.