Detection of green fluorescent protein in a single bacterium by capillary electrophoresis with laser-induced fluorescence.
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.
Turner
Emily H
EH
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
Lauterbach
Kevin
K
Pugsley
Haley R
HR
Palmer
Vanessa R
VR
Dovichi
Norman J
NJ
D034301:Deinococcus / Q000737:chemistry* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D007834:Lasers
D049452:Green Fluorescent Proteins