Difference between revisions of "Part:BBa K678004"

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===Contribution: NUDT_CHINA 2019===
 
===Contribution: NUDT_CHINA 2019===
  
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Summary：in this part, we made contributions to the quantitative characterization data of mammalian PGK Promoter. We measured the strength of this promoter by the expression of firefly luciferase at different time.  
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Summary：in this part, we made contributions to the quantitative characterization data of mammalian PGK Promoter. We measured the strength of this promoter by the expression of firefly luciferase reporter gene in HepG2 cells.  
  
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Characterization: To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, pmirGLO carrying PGK promoter was transfected into liver carcinoma cell line HepG2. 0, 6, 12 and 24h after transfection, cells were harvested, and firefly luciferase activity was measured by Beyotime™  Dual Luciferase Reporter Gene Assay Kit. Results are shown below.  
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==Methods==
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To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, plasmids carrying PGK promoter-firefly luciferase cassette was transfected into liver carcinoma cell line HepG2 by lipo3000 under standard proytocol. Cells were cultures under standard DMEM High glucose medium under 5% CO2 and 37 ℃. Cells were harvested 0, 6, 12 and 24h after transfection, and firefly luciferase activity was measured by Beyotime™  Dual Luciferase Reporter Gene Assay Kit.
  
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https://static.igem.org/mediawiki/parts/b/b3/T--NUDT_CHINA--PGK2.jpg
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==Results==
  
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The result showed a significant increase of firefly luciferase within 24 hours of transfection.
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https://static.igem.org/mediawiki/parts/b/b3/T--NUDT_CHINA--PGK2.jpg
  
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Figure 1. PGK-drived firefly luciferase activity.
 
  
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The result showed that within 24h, transcription of the mammalian PGK promoter-drived luciferase level keeps rising and reached maximum at 24h after transfection.  
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Figure 1. PGK-drived firefly luciferase activity, Error bar represents SD of at least 3 biological replicates.
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Revision as of 01:37, 22 October 2019


PGK, mammalian promoter

Promoter for expression of genes in mammalian cells

Contribution: NUDT_CHINA 2019

Summary：in this part, we made contributions to the quantitative characterization data of mammalian PGK Promoter. We measured the strength of this promoter by the expression of firefly luciferase reporter gene in HepG2 cells.

Methods

To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, plasmids carrying PGK promoter-firefly luciferase cassette was transfected into liver carcinoma cell line HepG2 by lipo3000 under standard proytocol. Cells were cultures under standard DMEM High glucose medium under 5% CO2 and 37 ℃. Cells were harvested 0, 6, 12 and 24h after transfection, and firefly luciferase activity was measured by Beyotime™ Dual Luciferase Reporter Gene Assay Kit.

Results

The result showed a significant increase of firefly luciferase within 24 hours of transfection.

T--NUDT_CHINA--PGK2.jpg


Figure 1. PGK-drived firefly luciferase activity, Error bar represents SD of at least 3 biological replicates.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 143
    Illegal PstI site found at 400
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 143
    Illegal PstI site found at 400
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 143
    Illegal PstI site found at 400
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 143
    Illegal PstI site found at 400
  • 1000
    COMPATIBLE WITH RFC[1000]