Difference between revisions of "Part:BBa K2440005"

Revision as of 18:45, 21 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

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: 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. Technically, SV40 promoter-carrying pmirGLO plasmid was transfected into liver carcinoma cell line HepG2. 0, 6, 12 and 24h after transfection, cells were harvested, and renilla luciferase activity was measured by Beyotime™ Dual Luciferase Reporter Gene Assay Kit. Results are shown below.

Figure 1. Renilla luciferase activity of pmiRGLO-tranfected HepG2 cells.

Results indicate that renilla luciferase expression keeps increasing with time and reached maximum after transfection of 24h.

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

@@ Line 23: / Line 23: @@
 ===Contribution:NUDT_CHINA, 2019===
-Summary: This year we added quantitative data the SV40 promoter.
+Summary: 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 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.
+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. Technically, SV40 promoter-carrying pmirGLO plasmid was transfected into liver carcinoma cell line HepG2. 0, 6, 12 and 24h after transfection, cells were harvested, and renilla luciferase activity was measured by Beyotime™ Dual Luciferase Reporter Gene Assay Kit. Results are shown below.
 https://2019.igem.org/wiki/images/5/5b/T--NUDT_CHINA--SV40.jpg
-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.
+Figure 1. Renilla luciferase activity of pmiRGLO-tranfected HepG2 cells.
 Results indicate that renilla luciferase expression keeps increasing with time and reached maximum after transfection of 24h.