High-throughput single-cell analysis of low copy number β-galactosidase by a laboratory-built high-sensitivity flow cytometer

被引：12

作者：

Yang, Lingling ^{[1
]}

Huang, Tianxun ^{[1
]}

Zhu, Shaobin ^{[1
]}

Zhou, Yingxing ^{[1
]}

Jiang, Yunbin ^{[1
]}

Wang, Shuo ^{[1
]}

Chen, Yuqing ^{[1
]}

Wu, Lina ^{[1
]}

Yan, Xiaomei ^{[1
]}

机构：

[1] Xiamen Univ, Key Lab Chem Biol Fujian Prov, Key Lab Analyt Sci, Dept Chem Biol,Coll Chem & Chem Engn, Xiamen 361005, Fujian, Peoples R China

来源：

BIOSENSORS & BIOELECTRONICS | 2013年 / 48卷

基金：

中国国家自然科学基金;

关键词：

Flow cytometry; Protein expression; Gene expression; beta-galactosidase; Low copy number; Single-cell analysis; ESCHERICHIA-COLI; GENE-EXPRESSION; PROTEIN EXPRESSION; QUANTIFICATION; NOISE; ABSOLUTE; LIMIT;

D O I：

10.1016/j.bios.2013.03.078

中图分类号：

Q6 [生物物理学];

学科分类号：

071011 ;

摘要：

Single-cell analysis is vital in providing insights into the heterogeneity in molecular content and phenotypic characteristics of complex or clonal cell populations. As many essential proteins and most transcription factors are produced at a low copy number, analytical tools with superior sensitivity to enable the analysis of low abundance proteins in single cells are in high demand. beta-galactosidase (beta-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Here we report the development of a high-throughput method for the single-cell analysis of low copy number beta-gal proteins using a laboratory-built high-sensitivity flow cytometer (HSFCM). Upon fluorescence staining with a fluorogenic substrate, quantitative measurements of the basal and near-basal expression of beta-gal in single Escherichia coli BL21(DE3) cells were demonstrated. Statistical distribution can be determined quickly by analyzing thousands of individual cells in 1-2 min, which reveals the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. Combined with the quantitative fluorometric assay and the rapid bacterial enumeration by HSFCM, the beta-gal expression distribution profile could be converted from arbitrary fluorescence units to protein copy numbers per cell. The sensitivity and speed of the HSFCM offers great capability in quantitative analysis of low abundance proteins in single cells, which would help gaining a deeper insight into the heterogeneity and fundamental biological processes in microbial populations. (C) 2013 Elsevier B.V. All rights reserved.

引用

页码：49 / 55

页数：7

共 42 条

[41] Progress in the development of techniques based on light scattering for single nanoparticle detection [J].