Team:Bielefeld-CeBiTec/Results/toolbox/photolysis

Photolysis

Design of 2-NPA-RS

At first, we designed a gene synthesis at IDT based on the selection experiment from Peters et al. (2009) to get an aminoacyl-tRNA-synthetase to incorpate 2-nitrophenylalanine. An alignment of the protein sequences of the native M. jannaschii TyrRS which was the basis of the selection experiment and the used 2-Nitrophenylalanine-synthetase is shown in Figure 1. They differ in ten amino acids.

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

We then used the protein sequence of the clone with the highest fidelity for 2-NPA and translated it into a gene sequence which was then codon optimized for E.coli. We designed it with matching overhangs of 35bp to a linearized ONBY-Part (K1416000) in pSB1C3 to get the sequence in a matching expression cassette. The psb1c3 backbone is a high copy plasmid and for an adequate usage of the aaRS it is needed on a low copy plasmid. We so used BioBrick assembly to get the insert of the new 2-NPA-Part (K2201200) in the low copy plasmid of pSB1K3 (Figure 2) for further use. The Insert of K2201200 in the low copy plasmid is available on request at the CeBiTec.

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

We also designed two fusion proteins to verify the incorporation and functionality of the 2-NPA (Figure 3). Plasmid I (K2201320) codes for a simple GFP-streptavidin fusion protein connected by a gly-gly-ser-linker. Plasmid II (K2201321) is homologous to plasmid I but has an amber codon in the middle of the linker. If transformed in E.coli BL21(DE3) (1) only the GFP-unit will be expressed (B). If cotransfromed with an aaRS for a noncanonical amino acid but without feeding the specific ncAA (2) the aaRS will incorporate other amino acids profoundly phenylalanine in the linker (C). If cotransformed and with the 2-NPA in the culture media (3) the fusion protein will be expressed with 2-NPA in the linker (D). The fusion protein can then be irradiated by light of a wavelength of 365nm (4) to induce the cleavage of the fusion protein to its GFP-unit (E) and the streptavidin-unit (F).

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

To proof the expression of the 2-NPA-RS in K2201200 we transformed this part, the ONBY-Part (K1416000) and a plasmid only containing the coding sequence of the fusion protein in E.coli BL21(DE3). The three transformants were cultivated in LB-Media for 16 hours at 37°C and 150 rpm. The cultures were harvested and the pellets lysed after the standard protocol of cell lysis in lysis buffer. Afterwards 15 µl of the samples were then transferred to an SDS-Page (Figure 4). There were two big bands present at approximately 35 kDA at the 2-NPA and the ONBY sample which correlates with the expected masses of the synthetases. We so are sure that the 2-NPA-RS in part K2201200 is well expressed when transformed in a matching E.coli strain like BL21(DE3).

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).