One of the ways to improve the signal to noise ratio of the reporter fluorescence, is to have a brighter (proportional to the extinction coefficient x quantum yield), more stable protein with a larger difference between the peak excitation and the emission wavelengths (Stoke’s shift) than the eGFP. Molecular Probes Handbook has a very nice section on fluorescence fundamentals, for more reading on how this all works.
With an improved Stoke’s shift, we can remove the excitation light from the emitted light collected by our light sensor without having very sophisticated filters, which can be expensive. Our current fluorescent reporter protein eGFP has an excitation wavelength of 488 nm and an emission wavelength of 509 nm which does not leave much room for the long-pass filter to filter out the excitation light source.
In addition, green fluorescence often has a higher background (autofluorescence) in biological systems. So moving away from this would be also beneficial.
In searching for such a protein, we found a really nice interactive fluorescent protein properties site by the UCSF Nikon Imaging Center. Looking for proteins with a large stokes shift, and brightness, we found the following proteins (extracted from the same site, pKa and other parameters are available there).
| Protein | λex | λem | EC | QY | Brightness | Maturation | References |
| (nm) | (nm) | (s) | (min) | ||||
| eGFP | 488 | 507 | 56000 | 0.6 | 33.6 | 25 | Yang 1996 |
| T-Sapphire | 399 | 511 | 44000 | 0.6 | 26.4 | ? | Zapata-Hommer 2003 |
| mAmetrine | 406 | 526 | 45000 | 0.58 | 26.1 | ? | Ai 2008 |
| LSSmOrange | 437 | 572 | 52000 | 0.45 | 23.4 | 138 | Shcherbakova 2012 |
After some discussion (look at the Shaner et al. reference below for the parameters to consider when choosing a fluorescent protein reporter), we decided to order the bacterial codon-optimized sequence for the protein LSSmOrange which has a large Stokes shift and that has a quantum yield and brightness that is not much lower than the one of eGFP. We also saw that LEDs were available in the wavelengths at relatively reasonable prices. The 430-440nm range seems to be used as plant grow lights and aquarium lights. We don’t know how the protein maturation time will impact the arsenic detection (more than 5x slower for the protein to express, fold correctly to fluoresce), but we will give it a go.
Original Relevant Papers
Shcherbakova, D. M., Hink, M. A., Joosen, L., Gadella, T. W. J., & Verkhusha, V. V. (2012). An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. Journal of the American Chemical Society, 134(18), 7913–7923. doi:10.1021/ja3018972
Pletnev, S., Shcherbakova, D. M., Subach, O. M., Pletneva, N. V., Malashkevich, V. N., Almo, S. C., et al. (2014). Orange fluorescent proteins: structural studies of LSSmOrange, PSmOrange and PSmOrange2. PLoS ONE, 9(6), e99136. doi:10.1371/journal.pone.0099136
Yang et al. Optimized Codon Usage and Chromophore Mutations Provide Enhanced Sensitivity with the Green Fluorescent Protein. Nucleic Acids Research 1996. 24(22): 4592-4593. doi: 10.1093/nar/24.22.4592
Zapata-Hommer et al. BMC Biotechnology 2003. 3(1): 5. doi: 10.1186/1472-6750-3-5
Ai et al. Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat Meth 2008. 5(5): 401-403. doi: 10.1038/nmeth.1207
A bit old, but a review useful to think about the parameters:
Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2(12), 905–909. doi:10.1038/nmeth819
biodesign for the real world 