Part:BBa_I712004

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 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

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).

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.