Study promoter activity using the Living Colors Fluorescent Timer, a fluorescent protein that shifts color from green to red over time (1). This color change provides a way to visualize the time frame of promoter activity, indicating where in an organism the promoter is active and also when it becomes inactive. Easily detect the red and green emissions indicating promoter activity with fluorescence microscopy or flow cytometry.
Easily Characterize Promoter Activity
The Fluorescent Timer is a mutant form of the DsRed fluorescent reporter, containing two amino acid substitutions which increase its fluorescence intensity and endow it with a distinct spectral property: as the Fluorescent Timer matures, it changes color—in a matter of hours, depending on the expression system used. Shortly after its synthesis, the Fluorescent Timer begins emitting green fluorescence but as time passes, the fluorophore undergoes additional changes that shift its fluorescence to longer wavelengths. When fully matured the protein is bright red. The protein’s color shift can be used to follow the on and off phases of gene expression (e.g., during embryogenesis and cell differentiation).
Fluorescent Timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo. The embryo was heat-shocked in a 33°C water bath. Promoter activity was studied during the heat shock recovery period. Green fluorescence was observed in the embryo as early as two hr into the recovery period. By 50 hr after heat shock, promoter activity had ceased, as indicated by the lack of green color.
pTimer (left) is primarily intended to serve as a convenient source of the Fluorescent Timer cDNA. Use pTimer-1 (right) to monitor transcription from different promoters and promoter/ enhancer combinations inserted into the MCS located upstream of the Fluorescent Timer coding sequence. Without the addition of a functional promoter, this vector will not express the Fluorescent Timer.
Detecting Timer Fluorescent Protein
You can detect the Fluorescent Timer with the DsRed Polyclonal Antibody.
You can use the DsRed1-C Sequencing Primer to sequence wild-type DsRed1 C-terminal gene fusions, including Timer fusions.
Terskikh, A., et al. (2000) Science290(5496):1585–1588.