Minimally invasive determination of mRNA concentration in single living bacteria Guet, Calin C ; https://orcid.org/0000-0001-6220-2052 Bruneaux, Luke Min, Taejin 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 similar to 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. Oxford University Press 2008 info:eu-repo/semantics/article doc-type:article text http://purl.org/coar/resource_type/c_2df8fbb1 https://research-explorer.ista.ac.at/record/3754 Guet CC, Bruneaux L, Min T, et al. Minimally invasive determination of mRNA concentration in single living bacteria. <i>Nucleic Acids Research</i>. 2008;36(12). doi:<a href="https://doi.org/10.1093/nar/gkn329">10.1093/nar/gkn329</a> eng info:eu-repo/semantics/altIdentifier/doi/10.1093/nar/gkn329 info:eu-repo/semantics/altIdentifier/issn/0305-1048 info:eu-repo/semantics/altIdentifier/e-issn/1362-4962 info:eu-repo/semantics/altIdentifier/pmid/18515347 https://creativecommons.org/licenses/by-nc/4.0/