Part:BBa_K2929004

mOrange + SpyTag

mOrange (BBa_E2050) conjugated to SpyTag (BBa_K1159201) via a 2xGGSG Linker. mOrange is a flourescent protein derived from RFP in Discosoma sp., and SpyTag is an oligopeptide which ligates to a SpyCatcher binding partner via an isopeptide bond. SpyTag is derived from the surface proteins of Streptococcus pyogenes.

This part is an improvement over BBa_E2050 as it is codon-optimised for Escherichia coli, and possesses a SpyTag which allows ligation to other proteins via a SpyCatcher domain. We do not predict the SpyTag domain to impact the expression or fluorescence of this part, so we believe BBa_K2929004 to be suitable reporter in E. coli.

Protein Mass: 26.7kDa Protein Excitation: 548nm Protein Emission: 562nm

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 820
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 594
  Illegal AgeI site found at 706
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization and improvement

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).

 

Results

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).

 

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.