mOrange + SpyTag
As our part improvement, we proposed codon-optimising the fluorescent protein BBa_E2050 mOrange for Escherichia coli, previously codon-optimised for Yeast. We also proposed adding the SpyTag biobrick BBa_K1159201 to mOrange to create a part with improved functionality, as the SpyTag would allow ligation to any SpyCatcher-conjugated part. We conjugated SpyTag to mOrange via a 2xGGSG linker then checked the tertiary structure using our RGN structural predictor. This predicted that SpyTag would be held distal to the mOrange protein (see figure 1), and thus that the SpyTag should be able to ligate with any SpyCatcher presented to the part, and that the SpyTag should not impact the fluorescence of the part.
Figure 1: Predicted tertiary structure of mOrange + SpyTag. mOrange is shown in green, joined via a linker to SpyTag (blue).
The full-length part was ordered from IDT where it was codon-optimised for E. coli. The part was then cloned into a pEHisTEV vector and expressed using E. coli BL21(DE3) cells (see methods). For comparison, yeast-optimised mOrange (BBa_E2050) was expressed analogously.
Figure 2 shows a small-scale Ni purification of mOrange+SpyTag. It shows bands of expressed protein at ~15kDa, approximately half the expected mass, ~30kDa. Mass spectrometry confirmed this band as being an RFP, hence we can establish this band is mOrange+SpyTag.
Figure 2: Small-scale Ni purification of mOrange+SpyTag. Lane 1 = Ladder, Lanes 2,6 = Unpurified sample, Lanes 3,7 = Flow-through sample, Lane 4,8 = Wash sample (5mM Imidazole), Lane 5,9 = Elution sample (250mM Imidazole).
Figure 3 shows a small-scale Ni purification of yeast-optimised mOrange. Bands at the expected mass 26.7kDa, as well as a visibly Orange purified solution (Figure 4) indicated correct expression of mOrange. However, purified bands at ~20kDa and 10kDa indicate cleavage at (at least) two sites in the protein (not one site as only the N-terminus has a His tag). As the purified mOrange+SpyTag solution was not visibly orange and at half the expected mass, we suggest that it too has been cleaved.
Figure 3: Small-scale Ni purification of mOrange. Lane 1 = Ladder, Lanes 2,6 = Unpurified sample, Lanes 3,7 = Flow-through sample, Lane 4,8 = Wash sample (5mM Imidazole), Lane 5,9 = Elution sample (250mM Imidazole).
Figure 4: The elution samples for mOrange, visibly orange.
To check the orange colour comes from the correct absorbance, we took an absorbance spectrum of the elution sample (Figure 5). We see a small peak at 548nm, the correct excitation wavelength for mOrange. Combining the correct expression weight and absorbance, we conclude successful expression of mOrange.
Figure 5: Absorbance spectrum for mOrange elution sample.
To assay the SpyTag activity of our new part we attempted to co-incubate the flow-through sample with a SpyCatcher analogue called OIPD (Open Isopeptide Domain). The rationale was that if mOrange+SpyTag was being cleaved in half, and the 6xHis-tagged N-terminal domain being retained on the column, then the C-terminal SpyTag should be present in the flow-through sample. Thus we set up a time course of co-incubation of the flow-through with OIPD to test if the C-terminal domain will ligate to the OIPD. We measured the mixture at 0, 30mins, 1hr, 2hr, 8hr, and overnight time intervals, then ran the samples on a gel (figure 6). This showed no clear ligation band, either being obscured by the OIPD dimer at 30kDa or just not visible. This suggests that the part protein may be being cleaved more than once, perhaps close to or in the SpyTag, preventing ligation to SpyCatcher or showing a negligible difference in mass.
Figure 6: Reaction of mOrange+SpyTag flow-through with OIPD over time. Lane 1 = Ladder, Lane 2 = OIPD, Lane 3 = mOrange+SpyTag flow-through, Lane 4 = Reaction mixture at 0hrs, Lane 5 = Reaction at 30mins, Lane 6 = reaction at 1hr, Lane 7 = reaction at 2hrs, Lane 8 = reaction and 8hrs, Lane 9 = reaction after overnight incubation.