Difference between revisions of "Part:BBa K2201342"

Latest revision as of 18:15, 25 October 2017

Fusion protein of RFP and GFP with an amber codon in the linker under T7-promotor control

This 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 added an T7-promotor to regulate the expression more precisely.

First we characterized the first translated units of the aaRS-test system by transforming them solely in E.coli BL21(DE3) without any aaRS. So only the RFP of this Texas part and the CFP of our improved part was expressed. Figure 1 shows six biological replicates of E.coli BL21(DE3), transformed with this part.

Figure 1: Six biological replicates of the CFP-YFP part BBa_K2201343 (top, greenish) and the RFP-GFP part (bottom, reddish).

Figure 2 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 maximum at 590 nm was used by Texas to excite the RFP and measure its emission on 610 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 2: 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).

We compared the variation of the emission signals of the test system when cotransformed with the CouAA-RS (BBa_K2201204) 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 3 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 3: 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).

Sequence and Features