Difference between revisions of "Part:BBa I712004"

Revision as of 09:17, 20 October 2009

CMV promoter

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

Sequence and Features

Functional Parameters

[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 thing 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)

Figure 1: Strength of the CMV promoter in different cell lines. [http://2009.igem.org/Team:Heidelberg/Project_Measurement Relative Expression Units (REU)] was determined by Flow cytometry; in HeLa and MCF-7 cells, this measurement was confirmed by an independent technique (Image analysis). 3 experiments on different days, three replicates for each measurement.

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. 3: Real-time RT-PCR data of cmv promoter. One group of HeLa cells were transfected with plasmid containing CMV promoter coupled to GFP. Another group with JeT promoter coupled to GFP served as reference. RNA was extracted after 20 h and 50 h, followed by the 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 total RNA, and then divided by total RNA generated by JET to obtain [http://2009.igem.org/Team:Heidelberg/Project_Measurement RMPU] .