A curve fitting method of quantifying green fluorescent protein expression level in Escherichia coli.

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.