Part:BBa_K2557016
TetO-miniCMV promoter-TP901
The TetO sequence, SV40 NLS, flag tag, miniCMV promoter, and TP901 recombinase are ligated in sequence to 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 part can convert the input signal of the upstream TEV into the output signal of the downstream mCherry. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of TP901 recombinase is turned on. Finally, TP901 functions as a recombinase to initiate expression of mCherry.
Characterization
TetR can effectively repress Tet operator in HEK 293T
Fig. 1 Inhibition of TetR on promoter with TetO sequence (A)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence (B)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.
TP901 works well in HEK 293T
Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells (A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments (B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. (C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.
The results of image B show that the reverse efficiency of TP901 recombinase is lower than Bxb1 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.
Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells.
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
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Illegal PstI site found at 964
Illegal PstI site found at 1675 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 964
Illegal PstI site found at 1675 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 986
Illegal BamHI site found at 325
Illegal BamHI site found at 1930 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 391
Illegal PstI site found at 964
Illegal PstI site found at 1675 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 391
Illegal PstI site found at 964
Illegal PstI site found at 1675
Illegal NgoMIV site found at 430
Illegal NgoMIV site found at 574
Illegal AgeI site found at 331 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1183
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