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To proof that our 2-NPA-RS is able to incorporate amino acids to the amber codon the parts was cotransformed with plasmid II (K2201321) in E.coli BL21(DE3) along with plasmid I (K2201320) and plasmid II was also transformed separately. After cultivation and cell lysis as mentioned above (in which one culture of the cotransformants was cultivated with 1mM and another without 2-NPA) the samples were transferred on an SDS-Page and a western-blot with anti-GFP-antibodies was performed (Figure 5). | To proof that our 2-NPA-RS is able to incorporate amino acids to the amber codon the parts was cotransformed with plasmid II (K2201321) in E.coli BL21(DE3) along with plasmid I (K2201320) and plasmid II was also transformed separately. After cultivation and cell lysis as mentioned above (in which one culture of the cotransformants was cultivated with 1mM and another without 2-NPA) the samples were transferred on an SDS-Page and a western-blot with anti-GFP-antibodies was performed (Figure 5). | ||
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/2/27/T--Bielefeld-CeBiTec--YKE_westernblot_results1.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/27/T--Bielefeld-CeBiTec--YKE_westernblot_results1.png"> | ||
<p class="figure subtitle"><b>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 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.</b><p> | <p class="figure subtitle"><b>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 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.</b><p> |
Revision as of 15:27, 4 October 2017
Design of 2-NPA-RS
Figure 1: Alignment of the protein sequences of the M. jannaschii tyrosyl synthetase and the 2-Nitrophenylalanine synthetase designed by Peters et al.
Cloning of this NPA-RS in pSB1C3 and pSB3T5
Figure 2: Two Plasmids we created for our toolkit for the iGEM community. Left: 2-NPA-RS in the pSB1C3 high copy plasmid (K2201200). Right: 2-NPA-RS in the pSB3T5 low copy plasmid (available on request) at the CeBiTec.
Design of fusion protein
Figure 3: Design of two plasmids for fusion proteins. I) Plasmid (K2201320) for reference protein of GFP (green) a linker (purple) and streptavidin (yellow) (A). II) Plasmid (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 (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 AS at Amber-codon when cotransformed with NPA-RS
Figure 4: SDS-Page of the expressed 2-NPA-RS (left) from K2201200 with ONBY-RS from 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 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 Microwellplate by irradiation of 365nm
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 plates: Left: Extinction of the plates for light of the wavelengths from 300 to 450 nm. Right: Calculated light-permeability in % of the three tested plates. At 365 nm wavelength 87% of the light permits the black nunc plate, 80 % permits the transparent nunc plate and 76 % permits the transparent greiner plate.