Quantitative imaging of gene expression in individual bacterial cells by chemiluminescence.
Recent gene expression studies at the single bacterial cell level have primarily used green fluorescent protein (GFP) as the reporter. However, fluorescence monitoring has intrinsic limitations, such as GFP maturation time, high background, and photobleaching. To overcome those problems, we introduce the alternative approach of chemiluminescence (CL) detection with firefly luciferase as the probe. Firefly luciferase is roughly 100 times more efficient and is faster in generating CL than bacterial luciferase but requires the introduction of luciferin, a species that is not native to bacteria. The difficulty of luciferin diffusion into the cells was solved by making use of cell membrane leakage during bacteria dehydration. In this scheme, the overall sensitivity of the system approaches the single protein molecule level. Quantitative studies of gene expression in BL21 and XLU102 bacteria can thus be performed.
Zhang
Yun
Y
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Phillips
Gregory J
GJ
Yeung
Edward S
ES
D001419:Bacteria / Q000166:cytology / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D049453:Luciferases, Bacterial / Q000235:genetics; D049409:Luciferases, Firefly / Q000235:genetics; D049449:Luminescence; D049408:Luminescent Agents / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods*; D013997:Time Factors
D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D049453:Luciferases, Bacterial