Design of the 2-Nitrophenylalanine-tRNA Synthetase
Figure 1: Alignment of the amino acids sequences with ClustalOmega of the M. jannaschii tyrosyl synthetase (M. jannaschii TryRS) and the 2-nitrophenylalanine synthetase (2-NPA-RS) designed by Peters et al., 2009.They differ in ten amino acids, which play a role in the binding process of the amino acid.
Cloning of the NPA-RS in pSB1C3 and pSB3T5
Figure 2: Two Plasmids we created for our toolbox. Left: 2-NPA-RS in the pSB1C3 high copy plasmid (BBa_K2201200). Right: 2-NPA-RS in the pSB3T5 low copy plasmid (available on request).
Design of the Fusion Protein
(1) Only the GFP-unit will be expressed (Figure 3B).
(2) If cotransformed with an aaRS for a non-canonical amino acid but without feeding the specific ncAA, the aaRS will incorporate other amino acids profoundly phenylalanine in the linker (Figure 3C).
(3) If cotransformed and with the 2-NPA in the culture media, the fusion protein will be expressed with 2-NPA in the linker (Figure 3D). The fusion protein can then be irradiated by light of a wavelength of < 367nm.
(4) This irradiation induces the cleavage of the fusion protein to its GFP-unit (Figure 3E) and the streptavidin-unit (Figure 3F).
Figure 3: Design of two plasmids for fusion proteins. I) Plasmid (BBa_K2201320) for reference protein of GFP (green) a linker (purple) and streptavidin (yellow) (A). II) Plasmid (BBa_K2201321) for the application protein with Amber-codon (black star) in the linker for three different protein variants after expression. 1: Solely expression leads to GFP-unit and linker to the Amber-codon (B). 2: Cotransformed with a 2-NPA-RS (BBa_K2201200) without 2-NPA leads to a fusion protein with an unspecific amino acid (presumably phenylalanine, red star) in the linker (C). 3: Cotransformed with 2-NPA-RS and 2-NPA leads to the functional fusion protein with 2-NPA (purple star) in the linker (D). 4: Irradiation of protein D leads to a cleavage of the fusion protein in the GFP unit (E) and the streptavidin unit (F).
Proof of Incorporation of Amino Acids at the Amber-Codon when Cotransformed with the 2-NPA-RS
Figure 4: SDS-Page of the expressed 2-NPA-RS (left) from BBa_K2201200 with ONBY-RS from BBa_K1416000 as positive control (middle) and the basic protein expression of BL21(DE3) as negative control (right).
Figure 5: Western blot with GFP-antibodies of the four different fusion proteins variants (Figure 3) as proof of the functionality of the 2-NPA-RS. The band marked with * in lane A is weak because of degradation of the fusion protein while the storage. The bands at approximately 45,0 kDa mark the mass of the whole fusion protein (~ 40,9 kDa), the bands at approximately 25,0 kDa mark the GFP-unit (~ 27,0 kDa) of the fusion protein.
Permeability of Microwellplates by Irradiation of 367nm
Figure 6: Three microwell plates tested for their suitability for the irradiation with the LED panel. Left: Black Nunc plate. Middle: Transparent Nunc plate. Right: Transparent Greiner plate.
Figure 7: Results of the irradiation test of the three microwell plate.
Change of Structure of 2-Nitrophenylalanine due to UV-Light
Figure 8: Changes in the absorption spectrum of 2-NPA in LB media while irradiated at 367 nm for 240 minutes. The emerging peak at ~ 340 nm indicates the change in the structure of 2-NPA from its native form, to the self-cyclized form.
Cleavage of the Fusion Protein
Figure 9: SDS-Page of four different samples. The whole fusion protein as positive control 1 (lane 1), GFP-unit as positive control 2 (lane 4), and two samples of the irradiated fusion protein containing 2-NPA after 1 hour (lane 2) and 5 hours (lane 3) of irradiation with UV-light. Highlighted in dark green are the bands of the whole fusion protein, in purple the bands of the 2-NPA-RS, in light green the GFP-unit of the fusion protein and in yellow the bands of the cleaved streptavidin-tag.
Figure 10: Western Blot of a SDS-Page similar to Figure 9. Anti-GFP antibodies were used to determine the bands of the SDS-Page. The blot proves that the low bands in Figure 9 are indeed cleaved streptavidin-Tags.
Figure 11: Scores 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.