Team:BM-AMU/Contribution

Team:BM-AMU- 2019.igem.org

Contribution



We found that the CAG promoter (BBa_K2217013) of AFCM-Egypt and the EF1a promoter (BBa_K2557033) of NAU-CHINA had no quantitative data until now. Therefore, we planned to measure the strength of CAG promoter (pCAG) , EF1a promoter (pEF1a) and compare them with other two promoters: CMV promoter (pCMV) and Ubiquitin promoter (pUbiquitin) to make contributions to the quantitative characterization data of pCAG and pEF1a.

Our team built four kinds of plasmids: which are built based on pGuide, with the pCAG, pCMV, pEF1a, pUbiquitin activating the expression of GFP, and then the expression of GFP strength can reflect the promoter strength after building complete four plasmids. Transfect the plasmids respectively into 293 cells by liposome transfection, then we collected samples after 24/48 hours. Finally, comparing GFP expression of CAG promoter, CMV, EF1a, Ubiquitin in 293 cells by flow cytometry, we obtained the result of the diagram below:

Contribution
Fig. 1 the absolute fluorescence for four different promoters.

The measurement units are arbitrary units of fluorescence.

The experimental procedures used in this assay involved measuring fluorescence using Median Fluorescence Intensity . Thus, the absolute values are arbitrary units, and cannot be directly compared to other systems. Our experiment, however, does reveal the relative strength of the CAG promoter device as compared to three promoters.

Contribution
Fig. 2 relative promoter strength of CAG, comparing with CMV, Ubiquitin, EF1a

The fluorescence in 24h were weaker than that in 48h among all of these promoters.In 48h, pCMV had about 2-fold greater fluorescence than the pCAG. pUbiqutin had about 1.1-fold to 1.2-fold greater fluorescence than the pCAG. pEF1a had about 1.5-fold greater fluorescence than the pCAG. The measurement units are arbitrary units of fluorescence.