A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression.
We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.
Schultz
D W
DW
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347.
Yarus
M
M
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000235:genetics*; D013489:Suppression, Genetic; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D012343:RNA, Transfer; D008156:Luciferases; D001616:beta-Galactosidase