Difference between revisions of "Part:BBa K678004"

Revision as of 18:03, 21 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 at different time.

Characterization: To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of PGK 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 for measurement of luciferase activity. Firefly luciferase activity was measured by Beyotime™ Dual Luciferase Reporter Gene Assay Kit. Results are shown below.

Figure 1. PGK-drived firefly luciferase activity.

The result showed that within 24h, transcription of the mammalian PGK promoter-drived luciferase level keeps rising and reached maximum at 24h after transfection. Sequence and Features

Assembly Compatibility:

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

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 ===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 expression change of firefly luciferase at different time.
+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.
-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.
+Characterization: To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of PGK 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 for measurement of luciferase activity. Firefly luciferase activity was measured by Beyotime™  Dual Luciferase Reporter Gene Assay Kit. Results are shown below.
 https://static.igem.org/mediawiki/parts/b/b3/T--NUDT_CHINA--PGK2.jpg
-Figure 1. PGK-drived firefly luciferase RLU in HepG2 cell line after transfection and culturing for 0, 6, 12 and 24 hours.
+Figure 1. PGK-drived firefly luciferase activity.
-Conclusions drawn from the results that within 24h, initiation strength of the mammalian PGK promoter keeps rising and reached maximum at 24h after transfection.
+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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 <span class='h3bb'>Sequence and Features</span>