Background

Bxb1, also known as Bxb1 gp35, is an recombinase comeing from Mycobacterium Phage Bxb1 and can bind to specific attB/P sites to catalyze DNA recombination. It helps the phage to integrate its genome into bacterial genome naturally. By constructing the attB/P sites in different directions, Bxb1 gp35 can catalyze the recombination of DNA between their sites, leading to inversion when attB/P are in opposite directions and excision when attB/P are in the same directions. The resulting attL and attR sequences cannot be recognized and bound by integrases alone, so the state after integration is stable. Bxb1 gp35 is widely used to construct combinational logic gate and synthetic biology.

Function In our Genetic Circuit

In our genetic circuit, Bxb1 regulated by TetR. When the inhibition released by TEV protease, Bxb1 can bind to specific attB/P sites to catalyze DNA recombination, leading to the expression of RFP.

Characterization

Method: Co-transfected with six different numbers of plasmids containing recombinase genes(miniCMV-Bxb1 and miniCMV-TP901) and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co - transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. The experiment was repeated three times.

Result:

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.

In conclusion, Bxb1 works well in HEK 293T as expected, so we present Bxb1(BBa_K2557001) as our best basic part.