Team:Nanjing NFLS/Improve
Improve.
Go to "Parts" and "Improve" from bottom navigation bar.
info
CMV is known as the strongest promoter in mammalian cells and utilized widely in mammalian expression systems. There are four NF-kB binding sites on CMV. Based on the work of Team Ljubljana 2007 BBa_I712004, Team Nanjing_NFLS 2018 has constructed mutated CMV P2 (BBa_K2597003) by changing the second natural NF-kB binding site into high-affinity SELEX-selected artificial sequence GGGGATTCCC, which owns higher transcriptional activity. (Figure 1)
Schematic of wt and mut P2 CMV. Boxes represent the sequences and relative positions of kBs in CMV. P, sequence used to create mut CMV.
Evaluation of optimized promoter activity with EGFP
To evaluate the transcriptional activity of promoters in a visualized format, we used EGFP as a reporter gene. The pEGFP vectors cloned with wt CMV and optimized mut CMV P2 were used to transfect CHO and HepG2 cells. The results reveal that EGFP expression was strongly driven by the promoters in the two cells. (Figure 2)
Detection of promoter activity by using EGFP as reporter gene. The HepG2 and CHO cells were transfected with the pEGFP plasmids containing different promoters. Cells were photographed under the bright field (left) and the fluorescence (right) with a fluorescence microscopy at 200 magnification. WT, wt CMV; CON, control (cells transfected with no plasmid).
Evaluation of optimized promoters with multiple cells
To test if the optimized mut CMV P2 also had high transcriptional activity in various cells, we evaluated the transcriptional of wt CMV and mut CMV P2 in five different mammalian cells lines, including HepG2, HeLa, K562, CHO and 293T, by using the dual-luciferase reporter assay and the GLuc reporter assay.
The dual-luciferase reporter assay revealed mut CMV P2 always had stronger transcriptional activity than wt CMV in five detected cells while GLuc reporter assay indicated that mut CMV P2 consistently outperformed wt CMV.
Evaluation transcriptional activity of promoters with multiple cells. (A and B) Detection of transcriptional activity of promoters by using the Dual-Luciferase reporter assay (A) and GLuc reporter assay (B) at 24 h post transfection. RLA, relative luciferase activity; RC, reporter constructs; NC, negative control. Statistical significance: mut CMV versus wt CMV.
Through our work, we constructed mutated CMV by mutating the natural kBs site, which tested to show higher transcriptional activity than the wt CMV. We hope the aforementioned improvement and relevant results could facilitate the utilization of CMV for other iGEM teams.