Fusion protein of CFP and YFP with an amber codon in the linker under T7-promotor control
This part is an improved version of the pFRY plasmid (BBa_K1416004) and pRXG (BBa_K2020040) consisting 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.
Our team 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.
We started the characterization of the CFP-unit of our improved part for the aaRS-test system. Here we determined one clear absorption maximum at 430 nm and one clear emission maximum at 475 nm (Figure 2). The two maxima were 45 nm apart, which allowed an easy excitement and emission process without any noise or measurement of the irradiated light.
In the cotransformants of the CFP-YFP system and the CouAA-RS (BBa_K2201204) we could confirm our desired FRET-system. 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 3). 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 4 shows the emission specters of the test system when cotransfomred with three different aaRS cultivated without their specific ncAA. They differ from each other in form and location of the emission maximum. The Prk-RS (BBa_K2201201) has its emission maximum at 520 nm, so very close to the YFP-emission maximum. This indicates that the Prk-RS is relatively unspecific, and couples native amino acids on the amber trna, which leads to a high amount of the whole CFP-YFP fusion protein. The emission spectrum when cotransformed with the CouAA-RS also has its maximum near 525 nm, but is a bit shifted to the lower wavelengths and has increased values near 475 nm, which indicates that more solely CFP-units are present. The 2-NPA-RS (BBa_K2201200) leads to an emission spectrum where even a little peak at 475 nm is visible, which indicates that this aaRS produces the less amount of the whole fusion protein. We so showed, that different aaRS cotransformed with our improved test system lead to distinguishable emission specters. The ratio of CFP-signal (emission at 475 nm) to YFP-signal (emission at 525 nm) gives information about the ratio of solely CFP-units to whole CFP-YFP FRET system when excited at the CFP absorption maximum of 430 nm.
The emission specters when cotransformed with the Prk-RS show a clear and significant shift, when cultivated with and without Prk (Figure 5). 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.
By comparing the emission specters of our improved test system when cotransformed with different aaRS, the efficiency of the aaRS can be determined. Recording whole emission specters 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. We therefore defined the negative, the positive and the mean rank.
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.
Figure 6 shows that the Prk-RS has a medium to low negative rank of 0,66±0,11, which means that without Prk supplemented to the media, native amino acids are coupled to the amber tRNA commonly. The positive rank of 3,34±0,02 is quite good, thus it means a significant increase in the expression of the whole fusion protein. The mean rank is 2,02±0,07, which is, as far as we can assess, a good quality for further work.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 770
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 1360
Illegal AgeI site found at 1472
- 1000COMPATIBLE WITH RFC