The pFRY plasmid consists of an mRFP domain which is connected to an sfGFP domain by a linker sequence containing an amber stop codon. The expression of the plasmid results either in red fluorescence, or - if the ncAA is incorporated at the amber stop codon within the linker site - in both: red and green fluorescence. By comparison of the fluorescence levels it is possible to determine the incorporation efficiency of the generated synthetase variants.
We liked this idea very much but had a little trouble in using it at our own synthetases. We investigated that there were some issues with the choice of RFP and GFP for the system, like a high overlap of the absorption spectra and a low emission level. We decided to improve the part by using CFP and YFP, for they form a FRET system (Ma et al., 2014) which leads to a more accurate distinction between the partial (CFP) and the whole (CFP-YFP) expressed fusion protein.