title: Absolute quantification of gene expression in individual bacterial cells using two-photon fluctuation microscopy.
abstract: Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 μM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks.
authors: Centre de Biochimie Structurale, INSERM U554, CNRS UMR 5048, Université Montpellier 1 and 2, F-34090 Montpellier, France.
authors: Matthew L
authors: ML
authors: Ferguson
authors: Dominique
authors: D
authors: Le Coq
authors: Matthieu
authors: M
authors: Jules
authors: Stéphane
authors: S
authors: Aymerich
authors: Nathalie
authors: N
authors: Declerck
authors: Catherine A
authors: CA
authors: Royer
mesh_terms: D001412:Bacillus subtilis / Q000166:cytology* / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D057230:Limit of Detection; D008164:Luminescent Proteins / Q000235:genetics; D008853:Microscopy / Q000379:methods*; D017785:Photons
chemical_list: D008164:Luminescent Proteins; D007544:Isopropyl Thiogalactoside