Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.
Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.
Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.Show MeSH
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Mentions: We first designed and constructed green (GrLuc), yellow (YeLuc), and red (RdLuc) luciferase derived from beetles (Viviani et al., 1999a, b; Fujii et al., 2007). We fused an N-terminal nuclear-localization signal (NLS) to concentrate these luciferases into a smaller volume and spread the light signal across fewer pixels (Figure 1A). For comparison, we also tested commercial beetle luciferases (CBG99, CBR, FLuc) and a new marine luciferase (NLuc; Hall et al., 2012) from Promega (Madison, WI; Figure 1B). Each luciferase reporter was regulated by a methionine-repressible promoter (MET17) and integrated into the yeast genome either in single copy or multiple copies; see Materials and Methods. We measured the emission spectrum of our designed luciferases to confirm that they were consistent with their expected color; see Supplemental Figure S1.
Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.