Difference between revisions of "Team:BostonU/Description"

BBa_I712004

The 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 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.