Team:DTU-Denmark/Overview/Strategy

To measure promoter activity we needed to choose a reporter. Instead of using GFP or a similar fluorescent protein we chose to use an RNA reporter known as Spinach. Spinach is a non-coding RNA aptamer that fluoresces only after binding to a specific ligand. By using Spinach instead of a protein reporter we could eliminate the effects of different translational efficiencies and measure RNA concentrations directly.
Measuring the RNA concentration and the degradation rate, would allow us to calculate the rate of formation of RNA, i.e. the transcription activity.

To be able to correlate the measured fluorescence to actual RNA concentrations we needed a standard series. To do this we mixed excess Spinach with known concentrations of ligand, and argued that the fluorescence values measured could be directly translated to the values measured from excess ligand and limiting Spinach as found in vivo.