Part:BBa_K2557017

TetO-EF1α promoter-TP901

The TetO sequence, SV40 NLS, flag tag, EF1α promoter, and Tp901 recombinase are ligated in sequence, and participate in intracellular signal processing. According to the feedback of the digital model, the different strength promoters upstream of the recombinase and the recombination efficiency of the recombinase will affect the stability of the system. Therefore, in order to optimize our system, we used three promoters and three recombinases, hoping to find the optimal solution.

Sequence and Features

Usage and Biology

This composite part can transduce the input signal of the upstream TEV protease into the expression of TP901 recombinase, which can turn on the expression of RFP. Here, we characterized the combination of the EF1α promoter and TP901.

Characterization

Fig. 1 Fluorescence microscope observation of HEK 293T only transfect with plasmids containing promoters with tetO sequence.

Fig. 1 shows that the strength of the three promoters in HEK 293T cells is from strong to weak: Ubc > EF1 α > miniCMV.

Fig. 2 Fluorescence microscope observation of HEK 293T after transfecting with different plasmid combination.

Fig. 2 shows that the flipping effect of EF1α-TP901 is less than that of miniCMV-TP901, which is inconsistent with the strength of their promoter indicated by Fig. 1. We hypothesized that the expression of EF1α-TP901 was less than that of miniCMV-TP901 due to the complexity of eukaryotic gene expression.

References

1. Blechl, A., Lin, J., Shao, M., Thilmony, R. & Thomson, J. The Bxb1 Recombinase Mediates Site-Specific Deletion in Transgenic Wheat. Plant Mol. Biol. Report. 30, 1357–1366 (2012).

2. Rutherford, K. & Van Duyne, G. D. The ins and outs of serine integrase site-specific recombination. Curr. Opin. Struct. Biol. 24, 125–131 (2014).

3. Ramos, J. L. et al. The TetR Family of Transcriptional Repressors The TetR Family of Transcriptional Repressors. Microbiol. Mol. Biol. Rev. 69, 326–356 (2005).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 565
  Illegal PstI site found at 1138
  Illegal PstI site found at 1849
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1138
  Illegal PstI site found at 1849
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1160
  Illegal BamHI site found at 499
  Illegal BamHI site found at 2104
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 565
  Illegal PstI site found at 1138
  Illegal PstI site found at 1849
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 565
  Illegal PstI site found at 1138
  Illegal PstI site found at 1849
  Illegal NgoMIV site found at 604
  Illegal NgoMIV site found at 748
  Illegal AgeI site found at 505
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1357