Detection of green fluorescent protein in a single bacterium by capillary electrophoresis with laser-induced fluorescence
Detection of green fluorescent protein in a single bacterium by capillary electrophoresis with laser-induced fluorescence
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DOI：
10.1021/ac061778r
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发表时间：
2007-01-15
影响因子：
7.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Turner, Emily H.;Lauterbach, Kevin;Dovichi, Norman J.
绿色荧光蛋白（Green fluorescence protein， GFP）是一种常用的单细胞蛋白表达监测报告蛋白。然而，来自内源性成分的自身荧光可以掩盖来自GFP的信号，特别是在原核生物中低表达水平时。我们采用毛细管电泳激光诱导荧光分析绿色荧光蛋白在单个细菌中的表达。毛细管电泳将绿色荧光蛋白从细胞自身荧光成分中分离出来，减少了背景信号，提高了检测限。我们的系统提供了100 ymol（60拷贝）的GFP检测限。为了证明该仪器的性能，我们采用了一个在recA启动子控制下表达GFP的耐辐射球菌模型系统。我们报告了单一耐辐射球菌细菌中绿色荧光蛋白和自荧光成分的分辨率和检测。本文报道了第一个在耐辐射菌中表达绿色荧光蛋白的例子，并首次通过毛细管电泳在单个细菌中检测到绿色荧光蛋白。
Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis.