Team:Carnegie Mellon/Met-Overview


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The design of fluorogen-activated biosensors

Our synthetic reporters consist of a Spinach RNA reporter and a FAP protein reporter. By placing a promoter of interest immediately upstream of Spinach, we can measure a fluorescence signal from mRNA that is transcribed from the promoter. To measure protein expression levels, we placed a ribosomal binding site (RBS) and a FAP at 14 base-pairs downstream of the Spinach sequence to avoid a steric clash between the Spinach RNA secondary structure and the ribosome. This way, we can measure the expression levels of both RNA and proteins in each cell over time. Our modular construct allows the plug-and-play of promoters, hence allowing tremendous flexibility in the characterization of any promoters. This coupled system has several advantages over traditional systems that only measure protein levels through fluorescent molecules such as green fluorescent proteins (GFP), CFP, and YFP. Our reporters differ significantly from the classical fluorescent proteins in that they only fluoresce upon the binding of specific fluorogens (malachite green for the FAP reporter and DFHBI for the Spinach reporter). This construct allows us to address differences in RNA and protein expression dynamics. The plasmid map is shown here.

A simplified version of our construct is shown here.

The design of fluorescence spectra and measurements

To measure both RNA and protein levels simultaneously in a single cell, we need to design the fluorescence spectra such that the emission spectra do not overlap significantly. Therefore, we have chosen to use a FAP with Ex/Em=635/660. This FAP has a far-red emmision spectra that would be well-separated from any green fluorescent probes. For RNA measurements, we used a Spinach with Ex/Em=469/501. As shown by the following fluorescence spectra, the emission spectra of both FAP and Spinach do not overlap. Note, however that the excitation spectra have a small overlap, which may prove to be useful in FRET experiments. For our experiments, we did not mix dyes in wells with induced cells.

L5 FAP (MG) Excitation and Emission Spectra

Spinach (DFHBI) Excitation and Emission Spectra

In our experiments, fluorescence intensities were measured using 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).

Sequences of Spinach and FAP

The coding sequence of the Spinach RNA reporter is as follows1:

The coding sequence of the FAP protein reporter is as follows2:

Note: In our experiments, we used an improved version of the original L5 FAP that was published in Nature Biotechnology in 2008. The engineered version binds malachite green faster than L5 FAP. Due to unresolved issues of intellectual property, we could not deposit these reporters in the parts registry. However, we may be able to share the parts upon formal requests by other labs.

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