Part:BBa_K4202003

This part is a fusion protein of mRFP and a engineered protein named SpyCatcher

SpyCatcher-mRFP is a self-designed fusion protein. mRFP is a engineered fluorescent protein. Thus, it`s N and C terminus are allowed to link to other proteins. So we connect SpyCatcher to theN terminus of mRFP through GS sequence to construct SpyCatcher-mRFP. In addition, the N-terminal fusion protein with 6*His tag can be used for protein purification and other experiments.

In our experiment, we hope to use this part and our another part (BBa_K4202004) to verify the feasibility of SpyCatcher-SpyTag system by protein experiments. The protein was also used for experimental validation of the biological scaffold module of our experiment in which we used fluorescence microscopy to characterize the module.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Result:

The plasmid PET-28a(+)-SpyCatcher-mRFP was synthesized by Genscript. And the plasmid wai transferred into BL21(DE3).We set several experience to explore the proper conditions for protein expression. And we finally choose the 25 ^oC, 220rpm ,0.1mM IPTG, to induce protein.

Fig 1 The culture of BL21(DE3) with PET-28a(+)-SpyCatcher-mRFP

Then we purified this protein by His-tag Protein Purification Kit.

Fig 2 The red resin during the purification experience

Fig 3 SDS-PAGE and Coomassie brilliantblue staining results of His-tag protein purification.The lanes have been noted

The SpyCatcher-mRFP and mRFP-SpyTag(BBa_K4202002) protein samples were diluted to appropriate fold. We collocted 200μl of each, mixed and added 100μl 5×PBS. The mixture was incubated at 37 ^oC for 2h. Sample was collected for SDS-PAGE and WesternBlot analysis.It`s worthy to note that we choose exhibit the whole PDVF to abtain a visual outcome.

Fig 1-2The result of Western Blot by two kinds of antibody . Spy:The mixture of two protein after incubation spy

We saw that in the lane of spy, we can clearly see the signal of Spycatcher-SpyTag complex and corresponding protein. However, in the lane of Spycatcher -mRFP and MRFP-SpyTag, we can only see the signal of the protein possessing the corresponding tag. So we can proof the function of SpyTag-SpyCatcher.