Difference between revisions of "Part:BBa K2557033"

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<b>Summary</b>：
 
<b>Summary</b>：
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BM-AMU 2019 made contributions to the quantitative characterization data of pEF1a. We measured the strength of EF1a promoter (pEF1a) and compare it with other three promoters.
 
BM-AMU 2019 made contributions to the quantitative characterization data of pEF1a. We measured the strength of EF1a promoter (pEF1a) and compare it with other three promoters.
 +
 
Our team built four kinds of plasmids which are built based on pGuide, with the pEF1a, pCAG, pCMV and pUbiquitin activating the expression of GFP. The ex-pression of GFP can reflect the promoter strength after building complete four plas-mids. Transfect the plasmids respectively into 293FT cells by liposome transfec-tion.The resistance of plasmids was used to screen the cells, then we collected samples after 24/48 hours. Finally, compare GFP expression of pEF1a, pCAG, pCMV and Ubiquitin in 293FT cells by flow cytometry. The experimental procedures used in this assay involved measuring fluorescence using Median Fluorescence Intensity . Our experiment, however, does reveal the relative strength of the EF1a  promoter device as compared to three promoters. we obtained the result of the diagram below:
 
Our team built four kinds of plasmids which are built based on pGuide, with the pEF1a, pCAG, pCMV and pUbiquitin activating the expression of GFP. The ex-pression of GFP can reflect the promoter strength after building complete four plas-mids. Transfect the plasmids respectively into 293FT cells by liposome transfec-tion.The resistance of plasmids was used to screen the cells, then we collected samples after 24/48 hours. Finally, compare GFP expression of pEF1a, pCAG, pCMV and Ubiquitin in 293FT cells by flow cytometry. The experimental procedures used in this assay involved measuring fluorescence using Median Fluorescence Intensity . Our experiment, however, does reveal the relative strength of the EF1a  promoter device as compared to three promoters. we obtained the result of the diagram below:
 
   
 
   
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[[Image:T--BM-AMU--part6.jpg|center|thumb|500px|'''Fig.2  relative promoter strength of EF1a, CAG, CMV and Ubiquitin. The fluorescence in 24h were weaker than that in 48h among all of these promoters. The measurement units are arbitrary units of fluorescence.''']]
 
[[Image:T--BM-AMU--part6.jpg|center|thumb|500px|'''Fig.2  relative promoter strength of EF1a, CAG, CMV and Ubiquitin. The fluorescence in 24h were weaker than that in 48h among all of these promoters. The measurement units are arbitrary units of fluorescence.''']]

Revision as of 01:18, 20 October 2019


EF1α promoter

The elongation factor-1 promoter is a high expression synthetic promoter in mammalian cells.

Characterization

Fig. 1 Fluorescence microscope observation of HEK 293T only transfect with plasmids containing promoters with tetO sequence.

Fig. 1 shows that the strength of the three promoters in HEK 293T cells is from strong to weak: Ubc > EF1 α > miniCMV.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution：BM-AMU 2019

CAG&EF1a

Summary：

BM-AMU 2019 made contributions to the quantitative characterization data of pEF1a. We measured the strength of EF1a promoter (pEF1a) and compare it with other three promoters.

Our team built four kinds of plasmids which are built based on pGuide, with the pEF1a, pCAG, pCMV and pUbiquitin activating the expression of GFP. The ex-pression of GFP can reflect the promoter strength after building complete four plas-mids. Transfect the plasmids respectively into 293FT cells by liposome transfec-tion.The resistance of plasmids was used to screen the cells, then we collected samples after 24/48 hours. Finally, compare GFP expression of pEF1a, pCAG, pCMV and Ubiquitin in 293FT cells by flow cytometry. The experimental procedures used in this assay involved measuring fluorescence using Median Fluorescence Intensity . Our experiment, however, does reveal the relative strength of the EF1a promoter device as compared to three promoters. we obtained the result of the diagram below:

Fig.2 relative promoter strength of EF1a, CAG, CMV and Ubiquitin. The fluorescence in 24h were weaker than that in 48h among all of these promoters. The measurement units are arbitrary units of fluorescence.