Nir R, Yisraeli Y, Lamed R, Sahar E
Department of Biotechnology, Tel-Aviv University, Israel.
Appl Environ Microbiol. 1990 Dec;56(12):3861-6. doi: 10.1128/aem.56.12.3861-3866.1990.
We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.
我们描述了一种通过流式细胞术定量测量细菌和酵母中β-半乳糖苷酶（β-gal）水平的新方法，该方法能够根据表达的活性对活的微生物细胞进行分选并重新培养。该方法基于将单个细胞包裹在直径为20至30微米的琼脂糖微珠中，并使用荧光底物荧光素-二-β-D-吡喃半乳糖苷（FDG）分析所形成菌落（包含数百个细胞）的β-gal活性。三株含有不同水平β-gal的大肠杆菌菌株用作模型系统。发现每个微珠测量的平均荧光与相应菌株提取物中的酶水平之间具有高度相关性。尽管使用FDG需要使细胞通透化，但发现了这样的条件，即每个菌落的一小部分保持存活，而大部分酶暴露于底物。这使得微菌落能够以接近100%的效率进行分选和平板接种。通过从β-gal阳性和β-gal阴性大肠杆菌菌株的人工混合物中选择β-gal阳性细胞，证明了该技术的潜力。
