Part:BBa_K4202002

This part is a fusion protein of mRFP and a short peptide named SpyTag

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

In our experiment, we hope to use this part and another part (BBa_K4202003) 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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 391
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 391
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 391
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 391
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results:

The plasmid PET-28a(+)-mRFP-Spytag 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 lysate of BL21(DE3) with PET-28a(+)-mRFP-SpyTag

Then we purified the mRFP-SpyTag by immuno precipitation.

Fig 2 SDS-PAGE and Coomassie brilliantblue staining results of HA-tag protein purification. Lane 1: Bacterial lysate(Input); Lane 2: Magnetic separation of supernatant after adsorption; Lane 3:Elution with loading buffer.

The mRFP-SpyTag and SpyCatcher-mRFP(BBa_K4202003) 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.

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Fig 3The 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.