Difference between revisions of "Part:BBa K203113"

Revision as of 13:39, 19 October 2009

JeT proximal-CMV core; constitutive promoter

Constitutive promoter at approx. 0.5 REU, created by cloning the Jet Proximal promoter in front of the CMV core promoter. Demonstrates that promoter strength can be modified by swapping Core promoters. Contains a HinDIII site between core and proximal promoter for that purpose.

Usage and Biology

Mammalian promoters can be subdivided into several "domains". The core promoter is the binding site of the basal transcription machinery, i.e. RNA polymerase and associated factors. Core promoters differ in composition, but are more or less similar for most genes [1]. The main regulatory domain is the proximal promoter, which is where regulatory elements bind. It can be very large (4kb), meaning that some transcription factors regulate transcription despite being very far away from the RNA polymerase. If the core promoter prevents optimal binding of the basal transcription machinery, promoter strength varies without affecting regulation.

[1] Heintzman ND, Ren B. The gateway to transcription: identifying, characterizing and understanding promoters in the eukaryotic genome. Cellular and Molecular Life Science 64, 386-400 (2007).

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]

Functional Parameters

We characterized this promoter in Part:BBa_K203100 as a first application for its newly developed units of promoter strength in mammalian cells, [http://2009.igem.org/Team:Heidelberg/Project_Measurement Relative Expression Units (REU)]. We found it to have a strength of 0,63 REU in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HeLa HeLa cells], 0,58 REU in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#MCF-7 MCF-7 cells] and 0,56 REU (SEM = 1,52) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#U2-OS U2-OS cells] cells (Fig 1).

Figure 1: Strength of the CMV core - JeT proximal promoter in different cell lines. Relative Expression Units (REU) was determined by Flow cytometry; 3 experiments on different days, three replicates for each measurement.

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 ===Usage and Biology===
-Mammalian promoters can be subdivided into several "domains". The core promoter is the binding site of the basal transcription machinery, i.e. RNA polymerase and associated factors. Core promoters differ in composition, but are more or less similar for most genes (reviewed in [9]). The main regulatory domain is the proximal promoter, which is where regulatory elements bind. It can be very large (4kb), meaning that some transcription factors regulate transcription despite being very far away from the RNA polymerase. If the core promoter prevents optimal binding of the basal transcription machinery, promoter strength varies without affecting regulation.
+Mammalian promoters can be subdivided into several "domains". The core promoter is the binding site of the basal transcription machinery, i.e. RNA polymerase and associated factors. Core promoters differ in composition, but are more or less similar for most genes [1]. The main regulatory domain is the proximal promoter, which is where regulatory elements bind. It can be very large (4kb), meaning that some transcription factors regulate transcription despite being very far away from the RNA polymerase. If the core promoter prevents optimal binding of the basal transcription machinery, promoter strength varies without affecting regulation.
+[1] Heintzman ND, Ren B. The gateway to transcription: identifying, characterizing and understanding promoters in the eukaryotic genome. Cellular and Molecular Life Science 64, 386-400 (2007).
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 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K203113 SequenceAndFeatures</partinfo>
 ===Functional Parameters===