(Oxford University Press, 2008) Guet, Calin C.; Bruneaux, Luke Julien; Min, Taejin L.; Siegal-Gaskins, Dan; Figueroa, Israel; Emonet, Thierry; Cluzel, Philippe
Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in
real-time [mRNA] down to »40nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is
proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of
mRNA concentration in individual living bacteria.
