Fusion protein of CFP and YFP with an amber codon in the linker under T7-promotor control
Introduction
This part is an improved version of the pFRY plasmid (BBa_K1416004) and pRXG (BBa_K2020040) consisting of a 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.
[http://2017.igem.org/Team:Bielefeld-CeBiTec/Improve Our team] 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.
Properties
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Figure 1: 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).
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Figure 2: 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).
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Figure 3: 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).
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Figure 4: Emission spectrum of three biological replicates each of cotransformants of the improved synthetase-test system (BBa_K2201343) with the Prk-RS. Three replicates were cultivated with their specific ncAA and three without it to compare the resulting shift in the emission spectrum.
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Figure 5: 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.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 770
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1360 Illegal AgeI site found at 1472
