Minimally invasive determination of mRNA concentration in single living bacteria

Nucleic Acids Res. 2008 Jul;36(12):e73. doi: 10.1093/nar/gkn329. Epub 2008 May 30.

Abstract

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 approximately 40 nM (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.

Publication types

  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Escherichia coli / genetics
  • Fluorescent Dyes / analysis
  • Kinetics
  • Luminescent Proteins / analysis
  • Luminescent Proteins / genetics
  • Protein Biosynthesis
  • RNA, Bacterial / analysis*
  • RNA, Messenger / analysis*
  • Recombinant Fusion Proteins / analysis
  • Spectrometry, Fluorescence / methods*
  • Transcription, Genetic

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • RNA, Bacterial
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • red fluorescent protein