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

• Published in: Biosensors & bioelectronics 2013-10, Vol.48, p.49-55
• Main Authors: Yang, Lingling, Huang, Tianxun, Zhu, Shaobin, Zhou, Yingxing, Jiang, Yunbin, Wang, Shuo, Chen, Yuqing, Wu, Lina, Yan, Xiaomei
• Format: Article
• Language: English
• Subjects: beta-Galactosidase - analysis; beta-Galactosidase - genetics; Biological and medical sciences; Biotechnology; Equipment Design; Escherichia coli - enzymology; Escherichia coli - genetics; Flow cytometry; Flow Cytometry - instrumentation; Fundamental and applied biological sciences. Psychology; Gene Dosage; Gene Expression; Low copy number; Protein expression; Single-cell analysis; Single-Cell Analysis - instrumentation; β-galactosidase
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2013.03.078

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. β-galactosidase (β-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 β-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 β-gal in single Escherichia coli BL21(DE3) cells were demonstrated. Statistical distribution can be determined quickly by analyzing thousands of individual cells in 1–2min, 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 β-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. [Display omitted] •Absolute quantification of low copy number β-gal in single bacterial cells is reported.•Distributions of basal and near-basal expression of β-gal were obtained in high-throughput.•This method was developed upon a laboratory-built high sensitivity flow cytometer.•This method takes advantage of the good cell retention of fluorogenic C12FDG.