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 expression change 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 at different time, we used firefly luciferase as its reporter gene. For method, we transfected equal quantity of pmirGLO into liver carcinoma cell line HepG2. Cells were cultured in High Glucose Dulbecco's Modified Eagle Medium with 10% FBS and under condition of 37℃, 5% CO2. 0, 6, 12 and 24h after transfection, cells were dissociated for luciferases (both firefly and renilla). Data was collected by Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit and analyzed by ImageJ. 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 ℃ with 10% FBS. 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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Figure 1. PGK-drived firefly luciferase RLU in HepG2 cell line after transfection and culturing for 0, 6, 12 and 24 hours.  
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https://static.igem.org/mediawiki/parts/thumb/f/fd/T--NUDT_CHINA--2019_Firefly.png/303px-T--NUDT_CHINA--2019_Firefly.png
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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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Conclusions drawn from the results that within 24h, initiation strength of the mammalian PGK promoter keeps rising and reached maximum at 24h after transfection.
 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 02:22, 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 ℃ with 10% FBS. 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.

303px-T--NUDT_CHINA--2019_Firefly.png


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]