Team:BostonU/Description
Our team worked to improve the characterization of a CMV promoter, BBa_I712004, part from the iGEM Registry. This part originated from the Heidelberg 2009 iGEM team as a “constitutive” promoter that could be expressed in HeLa mammalian cells. The cytomegalovirus (CMV) promoter is commonly used for gene expression in mammalian cells.
To characterize the part, our team cloned it upstream of a GFP in the pSB1C3 backbone, transiently transfected the plasmid into HEK293FT cells using PEI-mediated transfection, and assayed expression through flow cytometry. The CMV promoter device successfully expressed GFP in HEK293FT cells.
As part of our characterization, this part was also directly compared to parts BBa_K1875016, and BBa_K1875018 created by our team as part of the Gemini Parts Library. Parts BBa_K1875016 and BBa_K1875018 are gRNA operator reporter vectors containing minimal CMV promoters driving expression of GFP. These new parts were co-transfected into HEK293FT cells with a dCas9-VPR expression vector and the corresponding gRNA expression vectors, and then assayed using flow cytometry. We measured fluorescence of the CMV promoter device relative to these devices.
Part BBa_K1875016, the operator containing only one binding site for the gRNA and dCas9-VPR complex, expressed GFP at a level lower than the CMV promoter. Part BBa_K1875018 , the operator containing three binding sites for the gRNA and dCas9-VPR complex, 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 expression systems unless re-tested in the same context. Our experiment, however, does reveal the relative strength of the CMV promoter device as compared to both of our well-characterized parts.
