Minimally invasive determination of mRNA concentration in single living bacteria.
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.
The James Franck Institute, Institute for Biophysical Dynamics, Department of Physics, The University of Chicago, 929 E 57th St, Chicago, IL 60637, USA.
Călin C
CC
Guet
Luke
L
Bruneaux
Taejin L
TL
Min
Dan
D
Siegal-Gaskins
Israel
I
Figueroa
Thierry
T
Emonet
Philippe
P
Cluzel
D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein