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===Contribution:NUDT_CHINA, 2019===
 
===Contribution:NUDT_CHINA, 2019===
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Summary: This year we added quantitative data the SV40 promoter.  
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Summary: Since SV40 promoter has never been characterized in HepG2 cells, This year we added quantitative characterization data of the SV40 promoter.  
  
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Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators[1]. To quantify the reliability of SV40 promoter characteristics with time, we made statistics of its downstream Renilla Luciferase expression as representation of SV40 activity at different time. We transferred 500ng plasmid of pmirGLO into each well of 24 well plate of HepG2 by Invitrogen Lipofectamine™3000. After transfection of 0h, 6h, 12h, 24h cells were gathered for test for Rluc expression. Data was collected by Beyotime™ RG027 Dual Luciferase Reporter Gene Assay Kit and analyzed by ImageJ. Results are shown below.
 
  
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https://2019.igem.org/wiki/images/5/5b/T--NUDT_CHINA--SV40.jpg
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Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators[1]. To characterize the SV40 promoter, renilla luciferase was used as the reporter gene to quantify the transcriptional strength.  
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Figure 1. SV40 promoter characteristics represented by renilla luciferase expression from HepG2 cells growing in High Glucose Dulbecco's Modified Eagle Medium with 10% FBS.  
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Results indicate that renilla luciferase expression keeps increasing with time and reached maximum after transfection of 24h.
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==Methods==
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To characterize the SV40 promoter, renilla luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, plasmids carrying sv40 promoter-renilla 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 renilla luciferase activity was measured by Beyotime™  Dual Luciferase Reporter Gene Assay Kit.
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==Results==
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Results showed significant increase of luciferase level within the first 24 hours of expression.
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https://static.igem.org/mediawiki/parts/thumb/6/67/T--NUDT_CHINA--2019_Rluc.png/261px-T--NUDT_CHINA--2019_Rluc.png
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Figure 1. Renilla luciferase activity of SV40-rLuc tranfected HepG2 cells. Error bar represents SD of at least 3 biological replicates.
  
 
===Reference===
 
===Reference===
  
 
[1] Gagniuc P, Ionescu-Tirgoviste C. (Apr 2013)."Gene promoters show chromosome-specificity and reveal chromosome territories in humans" BMC Genomics. 14:278. PMID: 23617842 PMCID: PMC3668249 DOI: 10.1186/1471-2164-14-278
 
[1] Gagniuc P, Ionescu-Tirgoviste C. (Apr 2013)."Gene promoters show chromosome-specificity and reveal chromosome territories in humans" BMC Genomics. 14:278. PMID: 23617842 PMCID: PMC3668249 DOI: 10.1186/1471-2164-14-278

Latest revision as of 02:22, 22 October 2019


SV40 promoter

This is a promoter gene derived from SV40. It could initiate the transcription of downstream gene.

Usage and Biology

The promoter is a DNA region that initiates the transcription of a particular gene. The promoter is located upstream of the same strand at the transcription initiation site of a particular gene. 1

At pmirGLO Vector, SV40 early enhancer/promotor is located at 426–844 nucleotides, with a length of 419 nucleotides. Its role is to initiates the transcription of the hRluc-neo fusion protein coding region.

Sequence and Features

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:NUDT_CHINA, 2019

Summary: Since SV40 promoter has never been characterized in HepG2 cells, This year we added quantitative characterization data of the SV40 promoter.


Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators[1]. To characterize the SV40 promoter, renilla luciferase was used as the reporter gene to quantify the transcriptional strength.

Methods

To characterize the SV40 promoter, renilla luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, plasmids carrying sv40 promoter-renilla 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 renilla luciferase activity was measured by Beyotime™ Dual Luciferase Reporter Gene Assay Kit.

Results

Results showed significant increase of luciferase level within the first 24 hours of expression.

261px-T--NUDT_CHINA--2019_Rluc.png

Figure 1. Renilla luciferase activity of SV40-rLuc tranfected HepG2 cells. Error bar represents SD of at least 3 biological replicates.

Reference

[1] Gagniuc P, Ionescu-Tirgoviste C. (Apr 2013)."Gene promoters show chromosome-specificity and reveal chromosome territories in humans" BMC Genomics. 14:278. PMID: 23617842 PMCID: PMC3668249 DOI: 10.1186/1471-2164-14-278