Concept

The idea for the basis of promoter characterization is that our FAP (known as Ben) is a conditionally fluorescent protein. Similarly, our construct, known as Spinach is also conditionally fluorescent, but the two fluorescent constructs are not promiscuous. By placing the promoter of interest immediately upstream of Spinach, we get a fluorescence signal from the mRNA that is transcribed. To record protein fluoresence signals, we placed a RBS 14 base pairs downstream of the Spinach sequence to avoid a steric clash between the Spinach supramolecular structure and the ribosome. After the RBS, we cloned in our FAP so we can record protein levels over time as well as the RNA levels. This coupled system has several advantages over a traditional system, which only measures protein levels. This allows us to characterize more properties of any given promoter and address unpredicted behavior. The plasmid map is shown here.

A simplified version of our construct is shown here.

Fluorescence Readings

In our experiments, fluorescence intensities were measured with a Tecan SafireII. Given the absorption spectra from the published papers on the L5 FAP and Spinach, we were able to take excitation/emission measurements in our assay.

L5 FAP (MG) Excitation and Emission Spectra

Spinach (DFHBI) Excitation and Emission Spectra