Difference between revisions of "Part:BBa K1119006"

Revision as of 03:38, 22 October 2019

CMV promoter

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.

Issues with Previously Submitted CMV Promoters

The Part Registry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. However, according to the user reviews, team LMU-Munich 2010 stated that this part is not a CMV promoter but rather a long version of lacI gene for prokaryotes. In addition, there are five twins of this CMV promoter, including BBa_I712004, which was submitted by Ljubljana 2007 team and [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Different_core_promoters_result_in_different_expression_strength| characterized by Heidelberg 2009] team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). Since we could not identify any reliable CMV promoter from the Part Registry, we decided to build one for our constitutive glyoxylate shunt construct.

Caution: If this promoter is fused to a mammalian translation unit using RFC10, the 5'UTR would only have 6nt. If users encounter lower or no expression upon assembly, including extra DNA spacer sequences between the CMV promoter and the first ATG codon might help.

Characterization

In our characterization, CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108).

The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.

The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator (BBa_K648013) that does not contain the CMV promoter.

The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization/cmv detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.

Figure 1. CMV promoter drives expression of GFP. HEK cells transfected with pCMV-GFP gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10 microns

Egypt-AFCM Team Improvement

[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] tried to improve this part by Fusing CMV enhancer to CMV promoter in one composite part to enhance its transcription activity and was resubmitted at BBa_K2217024. Part characterization and usage can be found at Part Experience

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. Sequence and Features