The pFRY plasmid consists of an mRFP CDS which is connected to an sfGFP CDS 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.
Figure 1: Comparison of the RFP-GFP (BBa_K2020040) and the CFP-YFP (K22012343) system. A) shows the expression of the RFP unit of the fusion protein when not cotransformed with an aaRS. B) shows the expression of the complete RFP-GFP fusion protein when cotransformed with an aaRS. C) shows the expression of the CFP unit of the improved part when not cotransformed with an aaRS. D) shows the expression of the complete CFP-YFP fusion protein of the improved part when cotransformed with an aaRS.
First we compared the CFP and the RFP domain of the test system by transforming them into BL21(DE3) solely and performing absorption and emission measurements. We detected that the absorption and the emission maxima of the RFP are equal to the information of Texas 2014. Nevertheless, they are only 20 nm apart from each other, which lead to the first measurement problems. Furthermore we detected a second absorption maximum at 505 nm, not mentioned by Texas 2014. This excitation wave length was more suitable for RFP than 585 nm, but on the other hand very close to the absorption maximum of GFP (485 nm). As a result, heavy excitement and emission of RFP were observed as a byproduct of the GFP analysis. The absorption and emission maxima of CFP are ~ 45 nm apart and thereby very precisely differentiable. Nevertheless, we could detect that the emission signal of the CFP is approximately five times higher than that of RFP, if both of them are excited at their absorption maxima under comparable conditions. We could replicate these results in vitro and in vivo.
We demonstrated that our FRET system is functional. Excitation of the YFP-CFP fusion protein at wavelength specific for YFP excitation (475 nm) or CFP excitation (430 nm) both leads to strong YFP fluorescence signals. This means that the energy of the CFP emission is transferred to the YFP and thus the FRET system is working. Also there was still a clear CFP signal present at 475 nm which means that only the whole fusion protein of CFP and YFP forms the FRET system, and that solely CFP, where no ncAA was incorporated in the linker, still is present and detectable alongside with the FRET system.
By comparing the emission spectra of our improved test system when cotransformed with different aaRS, the efficiency of the aaRS was analyzed. Our results imply that a comparison of different aaRS is possible, just by comparison of the relative emission at 475 nm, representing the CFP amount, and at 525 nm, representing the fusion protein amount of the cotransformants when cultivated with and without the specific ncAA.
Therefore, we developed a ranking system for the evaluation of synthetases based on initial work by Texas 2014. This new scoring system is suitable for our improved FRET system due to the integration of a negative and a positive score.
RFP and CFP Activity of the Systems in vitro
We cultivated each transformant in 50 mL of LB-media in a 500 mL cultivation flask for 16 hours. Protein expression was induced with IPTG after 6 hours of cultivation at 37 °C and 150 rpm. We harvested the cells and lysated the cell pellet with the Ribolyzer and centrifuged the cell debris. Afterwards, we measured the fluorescence in 200 µL of the clear lysate.
Figure 1 shows the results of the absorption measurement of the RFP from wavelengths of 475 nm to 750 nm. We detected two absorption maxima at 505 nm and 590 nm. The absorption maximum at 590 nm was used by Texas to excite the RFP and measure its emission on 605 nm. Here we faced the first challenge. When exciting and measuring at the determined absorption and emission maxima, the received amount of the irradiated light was high, thus the measurement of the RFP-signal was impossible. To solve this problem we decreased the excitation wavelength by 5 nm to 585 nm and increased the emission wavelength by 5 nm to 615 nm. This way, the noise was completely removed and we could proceed the measurements.
To tackle this issue we decided to continue the measurements at the absorption maximum of 505 nm. This also leads to an excitement of the RFP and an emission maximum at 610 nm, but prevents the possibility of measuring the irradiated light.
Figure 1: Relative absorption and emission of RFP. The highest value equals one. The maximal absorption peaks at ~505 nm (grey) and at ~590 nm is another local maximum. The emission maximum lays at ~ 610 nm (red).
Figure 2: Relative absorption and emission of CFP. The highest value equals one. The maximal absorption lays at ~430 nm (grey line). The emission maximum lays at ~475 nm (cyan line).
Figure 3: Emission normalized to the amount of protein in the cell lysate of the two samples. The emisson of CFP after excitation at 430 nm is shown in cyan. RFP emission when excited at 505 nm is shown in red squares, when excited at 585 nm is shown in red triangles. The lower red course is caused by the emission of RFP when excited at 505 nm and the higher red course is caused by RFP when excited with 585 nm.
Figure 4: Maximal emission of CFP (cyan) and RFP (red) normalized to the amount of protein in the cell lysate. The samples were excited at the specific absorption maximum (CFP: 430, RFP: 505, 585).
RFP and CFP Activity of the Systems in vivo
Figure 5: Six biological replicates of E.coli expressing CFP-YFP or RFP-GFP. The CFP-YFP part BBa_K2201343 (top, greenish) and the RFP-GFP part BBa_K2020040 (bottom, reddish). The cells were cultivated in 1 mL LB-media for 24 hours at 30 °C with 400 rpm.The colors indicate that all cultures started expressing the desired fusion protein.
Figure 6: Maximal emission of CFP (cyan) and RFP (red) normalized to the OD600 of the six replicates. The samples were excited at the specific absorption maximum (CFP: 430, RFP: 505, 585).
GFP and YFP/FRET Activity of the Systems in vivo
We compared the variation of the emission signals of the test systems when cotransformed with the CouAA-RS to verify the production of the complete fusion proteins. This is possible due to the limited specificity and fidelity of artificially selected and evolved synthetases. They will always couple native amino acids to the amber tRNA and thus a proportion of the whole fusion protein will always be synthesized.
Figure 7 shows the emission spectrum of a culture of the cotransformants described above. When excited at the absorption maximum of GFP, at 485 nm, we can now measure a GFP-signal at 525 nm, which was not present when no CouAA-RS was present in the cells. Even when the GFP-signal was clear to see, and an expression of the whole RFP-GFP fusion protein was thereby confirmed, we also see a very high emission of RFP. This was caused by the broad overlap in the absorption spectrum of GFP and RFP. The RFP and GFP present in the cell will compete for the light. Therefore, excitation of this protein in the sample will lead to a weaker GFP-signal than would be expected, if no RFP would be present.
Figure 7: Relative emission spectrum (excited at 485 nm, gray) of the RFP-GFP system. Cotransformed with the CouAA-RS (BBa_K2201204), cultivated without CouAA. Maximal emission of the GFP at 525 nm (green line) and maximal emission of RFP at 610 nm (red line).
First, we excited the sample with light of 475 nm, which corresponds to the emission maximum of CFP and a reasonable absorption for YFP. The measured emission signal of the CFP-YFP system had a maximum at 525 nm (Figure 8). This matches with the expected YFP emission maximum. There was strong evidence that through the cotransformation, a specific amount of the whole CFP-YFP fusion protein was expressed in the cells. We then excited the sample at 430 nm, the absorption maximum of CFP. If there were no FRET system present in the sample we would expect a CFP signal with an emission maximum at 475 nm. The measured emission spectrum still had its maximum near 525 nm. We confirmed the FRET system by measuring the emission of light at the YFP emission maximum when excited at the CFP absorption maximum. This can only be done, if the energy of the excited CFP is transferred to the YFP before the emission process.
Figure 8: Relative emission spectrum (CFP signal: excited at 430 nm; YFP signal: excited at 475 nm) of the CFP-YFP system. Cotransformed with the CouAA-RS (BBa_K2201204). Maximal absorption of CFP at 430 nm (gray line). Maximal emission of CFP at 475 nm (blue). Maximal emission of YFP at 525 nm (yellow).
Comparison of Different aaRS Based on the CFP-YFP FRET-System
Figure 9: Relative emission spectrum (exited at 430 nm) of the CFP-YFP system (BBa_K2201343). Cotransformed with the CouAA-RS (BBa_K2201204), Prk-RS (BBa_K2201201) and the 2-NPA-RS (BBa_K2201200). All cultivated without their specific non-canonical amino acid. Maximal emission of CFP at 475 nm (blue). Maximal emission of YFP / FRET at 525 nm (yellow).
The emission spectra when cotransformed with the Prk-RS showed a clear and a substantial shift, when cultivated with and without Prk, respectively (Figure 10, left). Without Prk there is a bulge in the emission peak at 475 nm, due to the presence of solely CFP-units, where the synthesis of the CFP-YFP fusion protein stopped at the amber codon in the linker. The maximum of the emission spectrum is shifted towards the maximal YFP emission of 525 nm but not located there. If cultivated with Prk the bulge at 475 nm is very small and the emission maximum is at 525 nm. This means that by supplementing the specific ncAA to the cultivation media, the Prk-RS couples more amino acids to the amber tRNA. This leads to a higher expression of the whole fusion protein and indicates that the Prk-RS is a specific and efficient aaRS.
Figure 10: Emission spectrum of three biological replicates. Each of cotransformants of the improved synthetase-test system (BBa_K2201343) with the Prk-RS (BBa_K2201201) left and the 2-NPA-RS (BBa_K2201200) right. Three replicates were cultivated with their specific ncAA and three without it to compare the resulting shift in the emission spectrum.
By comparing the emission spectra of our improved test system when cotransformed with different aaRS, the efficiency of the aaRS can be determinated. Recording whole emission spestres is interesting but a bit time consuming if many aaRS should be testes for their quality. The results this far imply that a comparison of different aaRS is possible, just by comparison of the relative emission at 475 nm, representing the CFP amount, and at 525 nm, representing the fusion protein amount of the cotransformants when cultivated with and without the specific ncAA.
Negative and Positive Selectivity and Ranking System for Synthetases
The negative score is the quotient of the CFP-signal to the YFP-signal when the aaRS is cultivated without the specific ncAA. Negative score = (emission at 475 m)/(emission at 525 nm). When there is no supplemented ncAA, a very specific synthetase will not or seldom couple native amino acids to the amber tRNA. This would lead to a very high amount of CFP-units compared to the whole CFP-YFP fusion protein, resulting in a strong CFP-signal and a weak YFP-signal. The higher the negative score, the higher is the specificity of the aaRS. The maximal negative score should be around 2.0, which would correspond to solely CFP expression.
The positive score is the quotient of the YFP-signal to the CFP-signal when the aaRS is cultivated with the specific ncAA. Positive score = (emission at 525)/(emission at 475). When there is the specific ncAA is supplemented to the media, an efficient synthetase should couple the ncAA to the amber tRNA. This would lead to a very high amount of whole fusion protein compared to the solely CFP-units, resulting in a strong YFP signal including a strong contribution by the FRET-signal. The higher the positive score, the higher is the efficiency of the aaRS. The maximal positive score cannot be estimated yet, but based on our tests values between four and five seem to be plausible.
An advantage of our ranking system is, that the mean of the positive and the negative score can be used to merge the two values. This enables us to assign one specific mean score to one specific synthetase and thus to arrange tested synthetases after their quality.
The resulting scores of the tested Prk-RS and 2-NPA-RS are shown in Figure 11.
Figure 11: Scores resulting from the synthetase-test system. The negative score results from the emission quotient CFP(475 nm)/YFP(525 nm) when cultivated without the specific ncAA. The positive score results from the emission quotient YFP(525 nm)/CFP(475 nm) when cultivated with the specific ncAA. The mean rank allows the combination of the negative and the positive score to compare the efficiency of synthetases among each other.
The 2-NPA-RS has also a medium to low negative score of 0.73±0.07, which means it is a bit more specific than the Prk-RS. The positive score of 1.60±0.06 is very low, as it is not half as high as the positive score of the Prk-RS. This leads to a mean score of just 1.68±0.07.