Biomass conversion Laboratory of Tianjin University R&D Center for Petrochemical Technology, Tianjin University, Tianjin 300072, PR China.
J Microbiol Methods. 2010 Feb;80(2):172-7. doi: 10.1016/j.mimet.2009.12.003. Epub 2009 Dec 11.
Green fluorescent protein (GFP) has all the essential properties of a quantitative reporter protein and the fluorescence of GFP is a reliable and quantitative reporter of underlying differences in expression levels. However, GFP fluorescence does not increase with cell density in direct proportion because of its fluorescence quenching. And the fluorescence quenching is always ignored by most GFP fluorescence assays that provide a measure of average fluorescence intensity over an entire sample cell population. We now propose a novel method that accurately quantifies the fluorescence intensity of GFP expressed in Escherichia coli by a fluorescence spectrophotometer. In our method, a cell containing GFP was regarded as a fluorochrome and the data processing was essentially different from the previous methods. The experimental assay data were curve fitted to calibrate the mean fluorescence intensity of a cell population. Thus, the impact of fluorescence quenching caused by cell density was amended. Moreover, the mean fluorescence intensity of a cell population was roughly proportional to the fluorescence quantum efficiency, a property of a fluorochrome, so it can be used to evaluate the GFP expression level in a given cell population.
绿色荧光蛋白（GFP）具有定量报告蛋白的所有基本特性，GFP 的荧光是表达水平下潜在差异的可靠且定量的报告。然而，由于 GFP 的荧光猝灭，GFP 的荧光不会与细胞密度成正比地增加。并且大多数 GFP 荧光测定法都忽略了荧光猝灭，这些方法提供了整个样本细胞群体的平均荧光强度的测量值。我们现在提出了一种新方法，该方法使用荧光分光光度计准确地量化大肠杆菌中 GFP 的荧光强度。在我们的方法中，含有 GFP 的细胞被视为荧光染料，并且数据处理与以前的方法在本质上有所不同。实验测定数据被曲线拟合以校准细胞群体的平均荧光强度。因此，修正了由细胞密度引起的荧光猝灭的影响。此外，细胞群体的平均荧光强度与荧光量子效率大致成比例，荧光量子效率是荧光染料的特性，因此可以用于评估给定细胞群体中的 GFP 表达水平。
