Part:BBa_K2243012:Experience

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Applications of BBa_K2243012

2018 NAU-CHINA obtained the sequence of this part and requested the plasmid from them. Since our chassis organism is mammalian cell, we optimized the codon and then we identified BBa_K2557001 in detail in HEK 293T cells. Although Bxb1 works well in HEK 293T cells, we did not repeat the activity reported by Peking iGEM 2017 team in E. coli.

Bxb1 works well in HEK 293T cells

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.

We did not repeat the activity reported by Peking iGEM 2017 team in E. coli.

Pronuclear verification of recombinase

Fig. 3 A total of 9 single colonies were verified. Lane 1-9, recombinase expression plasmid validation; line 10, DL2000 DNA Marker; line 11-19, reporter gene expression plasmid validation;line 20, DL2000 DNA Marker.

Fig. 4 Two repetitions were selected and the results showed no obvious green fluorescence

Fig.5 Two replicates were selected after addition of the inducer and the results showed no obvious red fluorescence The result shows no obvious fluorescence. We changed some conditions, such as lowering the temperature, adjusting the rotation speed, adjusting the time, etc. But we still did not get the expected results. We consulted the teacher and the teacher replied that there might be weak fluorescence but our instrument couldn't detect it.

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