Team:NAU-CHINA/Composite Part

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PARTS

Composite Parts

Our Best Composite Part:BBa_K2557010

We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.

Function In Our Genetic Circuit

This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. 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 Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.

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.

Bxb1 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 Bxb1 recombinase is higher than 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. 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, so we present BBa_K2557010 as the Our Best Composite Part.