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 at their maximum excitation and emission peaks with a 10nm bandwidth and optimal gain (100 for Spinach and 255 for the FAP). FAP Ex/Em=635/660 and Spinach Ex/Em=469/501.

L5 FAP (MG) Excitation and Emission Spectra

Spinach (DFHBI) Excitation and Emission Spectra

Sequences

The Spinach sequence we used is as follows¹:
GCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGAGTAATTTACGTCGACGACGCAACCGAATGAAATGGTGAAG
GACGGGTCCAGGTGTGGCTGCTTCGGCAGTGCAGCTTGTTGAGTAGAGTGTGAGCTCCGTAACTGGTCGCGTCG
ACGTCGATGGTTGCGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGCGCCA
The L5 sequence is as follows²:
QAVVTQEPSVTVSPGGTVILTCGSSTGACTSGHYANWFQQKPGQAPRALIFETDKKYSWTPGRFSGSLLGAKAA
LTISDAQPEDEAEYYCSLSDVDGYLFGGGTQLTVLS
In our experiments, we used an engineered version of the original L5 FAP that was published in Nature Biotechnology in 2008. The engineered version is easier to express in E. coli than the original. For proprietary reasons, we are giving out the modified version of the L5 construct that has already been published by Shruti et al. Bear in mind that our construct is very similar to the sequence given above and should give other labs similar results, should they choose to use this technology.

¹RNA Mimics of Green Fluorescent Protein. Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey, et al. Science 29 July 2011: 333 (6042), 642-646. [DOI:10.1126/science.1207339]
²Shruti S, Urban-Ciecko J, Fitzpatrick JA, Brenner R, Bruchez MP, et al. (2012) The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression. PLoS ONE 7(3): e33429. doi:10.1371/journal.pone.0033429