Difference between revisions of "Part:BBa I712004"

Revision as of 19:33, 21 October 2009

CMV promoter

a constitutive expression promoter for use in mammalian cells. Ribosome binding site is included.

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

negative_regulators	-NA-
positive_regulators	-NA-

[http://2009.igem.org/Team:Heidelberg Heidelberg 2009 iGEM team] 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)] and [http://2009.igem.org/Team:Heidelberg/Project_Measurement Relative Mammalian Promoter Units (RMPU)], where RMPU is directly proportional to PoPs and measured on a RNA level, whereas REU is measured on the protein level. We found CMV to have a strength of 5,52 REU (Standard Error of the Mean = 0,60) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HeLa HeLa cells], 6,57 (SEM = 0,91) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#MCF-7 MCF-7 cells] and 9,96 (SEM = 1,52) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#U2-OS U2-OS cells] cells (Fig 1). We furthermore found that CMV is not a truly constitutive promoter (but we [http://2009.igem.org/Team:Heidelberg/Project_Measurement#There_are_no_truly_constitutive_promoters_in_mammalian_cells argue] that no such promoter exists). In [http://2009.igem.org/Team:Heidelberg/Project_Measurement RMPU] (a mRNA-based unit directly proportional to PoPs), CMV has an activity of approx. 2.89 RPMU in HeLa cells,as measured by qRT-PCR (Fig. 3)

Fig. 5: Strength of the CMV promoter in different cell lines in REU. [http://2009.igem.org/Team:Heidelberg/Project_Measurement Relative Expression Units (REU)] were determined by three independent flow cytometry measurements on three different days. This measurement was confirmed by an independent technique - image analysis.

Figure 2: CMV strength changes depending on conditions As [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Identification_of_challenges_unique_to_higher_eukaryotes there are no truly constitutive promoters in mammalian cells], both the reference promoter (Part:BBa_K203112) and CMV fluctuate dependent on condition (Erolimus induces extreme starvation). Measured by Flow cytometry 20 hours after transfection (unless specified otherwise).

Fig. 4: Real-time RT-PCR data of CMV and JeT promoters.One group of HeLa cells were transfected with plasmid containing CMV promoter coupled to GFP. Another group with JeT promoter coupled to GFP was used as reference. RNA was extracted after 20 h and 50 h, followed by real-time RT-PCR. The Ct values were collected with a threshold of 0.05. The CMV activity compared to JeT at the same time point was calculated in MatLab as "arbitrary units" which correspond to amount of mRNA.

Fig. 5: Real-time RT-PCR data of CMV promoter The RMPU was calculated with data from Fig. 4.

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-[[Image:Qpcr_result.png|center|350px|thumb|<div style="text-align:justify;">'''Fig. 6: Real-time RT-PCR data of CMV promoter'''. Twelve replicates of HeLa cells were transfected with a plasmid containing the CMV promoter coupled to GFP. Another set of twelve replicates was transfected with the JeT promoter coupled to GFP served as reference. Total amount of RNA generated by CMV was divided by the total amount of RNA generated by JeT to obtain [http://2009.igem.org/Team:Heidelberg/Project_Measurement RMPU]. </div>]]
+[[Image:qPCR_result_matlab.png|thumb|left|350px|<div style="text-align:justify;">'''Fig. 4: Real-time RT-PCR data of CMV and JeT promoters.'''One group of HeLa cells were transfected with plasmid containing CMV promoter coupled to GFP. Another group with JeT promoter coupled to GFP was used as reference. RNA was extracted after 20 h and 50 h, followed by real-time RT-PCR. The Ct values were collected with a threshold of 0.05. The CMV activity compared to JeT at the same time point was calculated in MatLab as "arbitrary units" which correspond to amount of mRNA.</div> ]]
+[[Image:qpcr_result.png|thumb|left|300px|<div style="text-align:justify;">'''Fig. 5: Real-time RT-PCR data of CMV promoter'''  The RMPU was calculated with data from Fig. 4.</div>]]
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