Difference between revisions of "Part:BBa I712004"

Revision as of 03:40, 22 October 2019

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 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. 1: 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. 3: 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. 4: Real-time RT-PCR data of CMV promoter Arbitrary units of CMV divided by that of JeT is the [http://2009.igem.org/Team:Heidelberg/Project_Measurement RMPU].

The [http://2016.igem.org/Team:BostonU/Description 2016 BostonU iGEM team] further characterized this CMV promoter part by cloning it upstream of a GFP, transiently transfecting in HEK293FT cells, and assaying expression through flow cytometry. The part was cloned upstream of a GFP gene in a pSB1C3 backbone and transiently transfected in HEK293FT cells using PEI-mediated transfection.

As the figure demonstrates, the pCMV had approximately 2-fold greater fluorescence than the single operator site plasmid from the Gemini Library; however, the triple operator site plasmid had about 3- to 4-fold greater fluorescence than the pCMV. The measurement units are arbitrary units of fluorescence.

As part of the characterization, this part was also directly compared to parts BBa_K1875016, and BBa_K1875018, created by the BostonU team as part of their project, Gemini. Parts BBa_K1875016 and BBa_K1875018 contain minimal CMV promoters and “guide operators” homologous to a 20 base pair guide RNA on a guide RNA expression vector. These new parts were co-transfected into HEK293FT cells with a dCas9-VPR and the complementary guide RNA expressing vector and then assayed using flow cytometry. Fluorescence of the CMV promoter device was measured relative to these devices.

The CMV promoter device successfully expressed GFP in HEK293FT cells. Part BBa_K1875016, the operator containing only one binding site for the dCas9-VPR, expressed GFP at a level lower than the CMV promoter while part BBa_K1875018 , the operator containing three binding sites, had higher GFP expression.

The experimental procedures used in this assay involved measuring fluorescence using Mean Fluorescence Intensity (M.F.I). Thus, the absolute values are arbitrary units, and cannot be directly compared to other systems. Our experiment, however, does reveal the relative strength of the CMV promoter device as compared to both of our well-characterized parts.

The single operator site plasmids from the Gemini Library had around the same fluorescence as the pCMV. The measurement units are arbitrary units of fluorescence.

The triple operator site plasmids from the Gemini Library had about 3- to 4-fold greater fluorescence than the pCMV. The measurement units are arbitrary units of fluorescence.

Improvement from iGEM 2018 Team Nanjing_NFLS

This year, we Nanjing_NFLS have improved the previous part BBa_I712004 by changing CMV’s second natural NF-kB binding site into high-affinity SELEX-selected artificial sequence GGGGATTCCC. We evaluated the optimized promoter activity with EGFP, and evaluated the promoter in various cells with the Dual-Luciferase reporter assay and Gluc reporter assay. The results revealed mut CMV BBa_K2597003 we created showed higher transcriptional activity compared to wt CMV. See more details here https://parts.igem.org/Part:BBa_K2597003

MIT-2019 Characterization

While the majority of our project was focused on engineering leader cells, we were also interested in manipulating the follower cells by genetically engineering HL-60 cells. We noticed that while there were several methods of transfection described for HL-60 cells ( including a project by the <a href=”http://2009.igem.org/Team:UCSF”>2009 UCSF iGEM team</a>, a paper by <a href=”http://limlab.ucsf.edu/papers/pdfs/park_2014.pdf”>Park et. al.</a>), we did not find any systematic data on the function of commonly used promoters in this cell type. Considering that HL-60 cells are relatively difficult to transfect and require harsh transfection conditions (electroporation) that can result in cell death and low transfection efficiency, we wanted to find a promoter that would lead to reliable and strong expression of transfected genes in order to facilitate our future experiments with the SynNotch system and engineering of leader cells to become followers.

In particular, we characterized the expression of the fluorescent proteins EYFP and TagBFP encoded on plasmids under the CMV and hEF1a promoters and transfected by electroporation into undifferentiated HL-60 cells.

Figure 1:

Figure 2a:

Figure 2b:

We observed a lot of cell death due to electroporation. Events from the flow cytometry analysis were first plotted on an FSC/SSC dot-plot graph to set an analysis gate, as shown in Figure 1. For the cells within the analyzed gate we looked at fluorescence in the FITC channel (excitation 488 nm, detection window 530/30 nm) for detection of EYFP and Pacific Blue channel (excitation 405 nm, detection window 450/50 nm) for detection of TagBFP. We found that 52% of cells transfected with CMV-EYFP were fluorescent in the yellow channel, and 35% of cells transfected with CMV-TagBFP were fluorescent in the blue channel. On the other hand, only 1.5% of cells transfected with hEF1a-EYFP and 8% of cells transfected with hEF1A-TagBFP were weakly fluorescent.

Figure 2 shows the overlay of histograms for untransfected cells (control, shown in green) and cells transfected with the fluorescent protein encoded under a CMV promoter (shown in blue) or hEF1a promoter (shown in red) for a) TagBFP and b) EYFP.