Team:Bielefeld-CeBiTec/Improve
Part Improvement
Introduction
The pFRY plasmid consists of an mRFP domain which is connected by a linker sequence containing an amber stop codon with a sfGFP domain. 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 fluorescence levels it is possible to determine 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 and decided to improve the part by using CFP and YFP, for they form a FRET system which leads to a more accurate distinction between the partial (CFP) and the whole (CFP-YFP) expressed fusion protein.
Short summary
First we compared the CFP and the RFP domain of the test system by transforming them into BL21(DE3) solely and performing some absorption and emission measurements. We detected that the absorption and the emission maxima of the RFP are equal to the information of Texas 2014 but they are only 20 nm apart, which lead to the first measurement problems. Furthermore we detected a second absorption maximum at 505 nm, not mentioned by Texas 2014. It was more suitable for exciting RFP but on the other hand very close to the absorption maximum of GFP (485 nm) which leads to a heavy excitement and emission of RFP when we wanted to excite only the GFP. The absorption and emission maxima of CFP are ~ 45 nm apart and so very sharp to differentiate. Also we could detect that the emission signal of the CFP is approximately five times higher than of RFP, if both of them are excited at their absorption maxima under comparable conditions. We could replicate there results in vitro and in vivo.
We proofed that our FRET system if functional, for there were high YFP-signals of the fusion protein detected, if excited at 475 nm, specific for YFP excitation, and when excited at 430 nm, specific for CFP excitation. This means that the energy of the CFP emission is transferred to the YFP and thus the FRET system is verified. Also there was a 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.
We so developed ne new ranking system for the quality of synthetases, slightly variated form the system of Texas 2014, matching for our improved FRET system.
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, with an induction 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. We then did the measurements with 200 µL of the clear lysate.
Figure 1 shows the results of the absorption mesuarement of the RFP from wavelengths of 475 nm to 750 nm. We detected two absorption maxima at 505 nm and 590 nm. The maximum at 590 nm was used by Texas to excite the RFP and measure its emission on 605 nm. Here we found the first problem. When we excited and measured at the determined absorption and emission maxima, the “Tecan Reader” received a high amount of the irradiated light, so that there was no measurement of the RFP-signal possible. 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. So there was no noise left and we could proceed the measurements, but not at the determined maxima.
To avoid this problem we decided to continue the measurements also at the absorption maximum of 505 nm, which also leads to an excitement of the RFP and an emission maximum at 610 nm.
Figure 1: Relative absorption and emission of RFP. The highest value equals one. The maximal absorption lays at ~505 nm (grey line) and at ~590 nm is another local maximum. The emission maximum lays at ~ 610 nm (red line).
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 (blue line).
Figure 3: Emission normalized to the amount of protein in the cell lysate of the two samples. The blue course is caused by the emission of CFP when excited at 430 nm. 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: The maximal emission of CFP (blue) and RFP (red) normalized to the amount of protein in the cell lysate when 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 the CFP-YFP part (top, greenish) and the RFP-GFP part (bottom, reddish).
Figure 6: The maximal emission of CFP (blue) and RFP (red) normalized to the OD600 of the six replicates when 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 whole fusion proteins. This is possible due to the limited specific and fidelity of artificial selected and evolved synthetase, so that the will also couple native amino acids to the amber tRNA and so some amount the whole fusion protein will be expressed.
In Figure 7 we see the emission spectrum of a culture of the cotransformants mentioned above. When excited at the absorption maximum of GFP, approximately 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 so an expression of the whole RFP-GFP fusion protein was confirmed, we also see a very high emission of RFP. This is caused by the high overlap in the absorption spectrum of GFP and RFP. The RFP and GFP present in the cell will so be in concurrence of the light, irradiated to excite the sample, which will lead to a weaker GFP-signal than there could be, if no RFP would be present.
Figure 7: Relative emission spectrum (excited at 485 nm, gray line) 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 good absorption property 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. We so were sure that through the cotransformation and 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. Gladly the measured emission spectrum still had its maximum near 525 nm, and so we confirmed the FRET system by proof the emission of light at the YFP emission maximum when excited at the CFP absorption maximum. This can only be when the energy of the CFP is transferred to the YFP bevor 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 line). Maximal emission of YFP at 525 nm (yellow line).
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 line). Maximal emission of YFP / FRET at 525 nm (yellow line).
The emission specters when cotransformed with the Prk-RS show a clear and significant shift, when cultivated with and without Prk (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 expression of the CFP-YFP fusion protein stopped at the amber codon in the linker. The maximum of the emission spectrum is shiftet 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 relatively 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 specters of our improved test system when cotransformed with different aaRS, the efficiency of the aaRS can be determinate. Recording whole emission spestres is interesting but a bit time consuming if many aaRS, for example after selection or modeling, should be tested. The results this far imply that a comparison of different aaRS is possible, just by compare 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 rank is the quotient of the CFP-signal to the YFP-signal when the aaRS is cultivated without the specific ncAA. Negative rank = (emission at 475 m)/(emission at 525 nm). We chose this value, because 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 rank, the higher is the specificity of the aaRS. The maximal negative rank should be around 2.0, which would correspond to solely CFP expression.
The positive rank is the quotient of the YFP-signal to the CFP-signal when the aaRS is cultivated with the specific ncAA. Positive rank = (emission 525)/(emission 475). We chose this value, because 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- / FRET-signal. The higher the positive rank, the higher is the efficiency of the aaRS. The maximal positive rank cannot be estimated yet, but based on our tests values to four or five seem to be plausible.
An advantage of our ranking system is, that the mean of the positive and the negative rank can be used to merge the two ranking values. This enables us to assign one specific mean rank to one specific synthetase and so to arrange tested synthetases after their quality.
The resulting ranks of the tested Prk-RS and 2-NPA-RS are shown in Figure 11.
Figure 11: Ranks resulting from the synthetase-test system. The negative rank results from the emission quotient CFP(475 nm)/YFP(525 nm) when cultivated without the specific ncAA. The positive rank 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 rank to compare the efficiency of synthetases among each other.
The 2-NPA-RS has also a medium to low negative rank of 0.73±0.07, which means it is a bit more specific than the Prk-RS. The positive rank of 1.60±0.06 is pretty bad, as it is not half as high as the positive rank of the Prk-RS. This leads to a mean rank of just 1.68±0.07.
