Difference between revisions of "Team:BostonU/Description"
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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.</p> | 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.</p> | ||
| − | <center><img src = "https://static.igem.org/mediawiki/2016 | + | <center><img src = "https://static.igem.org/mediawiki/2016/7/7a/T--BostonU--Bba_I712004_Validation.png" style = "padding:5px 150px 15px 150px; width:80%;"></center> |
<p style = "font-size:150%; padding:5px 150px 15px 150px; color:#0071A7;">BBa_I712004 compared to two Gemini operator promoter plasmids containing the same gRNA operator. The CMV fluoresced twice as bright as the single operator promoter plasmid (denoted by the singular operator site) and about a fourth as bright as the triple operator promoter plasmid (denoted by the triple operator site). </p> | <p style = "font-size:150%; padding:5px 150px 15px 150px; color:#0071A7;">BBa_I712004 compared to two Gemini operator promoter plasmids containing the same gRNA operator. The CMV fluoresced twice as bright as the single operator promoter plasmid (denoted by the singular operator site) and about a fourth as bright as the triple operator promoter plasmid (denoted by the triple operator site). </p> | ||
| − | <center><img src = "https://static.igem.org/mediawiki/2016 | + | <center><img src = "https://static.igem.org/mediawiki/2016/1/10/T--BostonU--Bba_I712004_Validation_Part2.png" style = "padding:5px 150px 15px 150px; width:80%;"></center> |
<p style = "font-size:150%; padding:5px 150px 15px 150px; color:#0071A7;"> | <p style = "font-size:150%; padding:5px 150px 15px 150px; color:#0071A7;"> | ||
CMV compared to three Gemini single operator promoters. The CMV expressed GFP at a greater intensity than all of the single operator promoters with the sole exception of the promoter containing the g3 operator. </p> | CMV compared to three Gemini single operator promoters. The CMV expressed GFP at a greater intensity than all of the single operator promoters with the sole exception of the promoter containing the g3 operator. </p> | ||
| − | <center><img src = "https://static.igem.org/mediawiki/2016 | + | <center><img src = "https://static.igem.org/mediawiki/2016/8/88/T--BostonU--Bba_I712004_Validation_Part3.png" style = "padding:5px 150px 15px 150px; width:80%;"></center> |
Revision as of 07:15, 16 October 2016
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.
As part of our characterization, this part was also directly compared to several of our Gemini library plasmids, more specifically: BBa_K1875014, BBa_K1875015, BBa_K1875016. 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.
Parts BBa_K1875014, BBa_K1875015, and BBa_K1875016 correspond to three of our single operator promoters driving GFP. In addition to these we compared three of our Gemini library plasmids with three multimerized operator sites upstream of a minimal promoter driving GFP. Flow cytometry found that five of the six Gemini parts we compared to the CMV fluoresced anywhere between half as bright to five times as bright as the CMV.
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.
BBa_I712004 compared to two Gemini operator promoter plasmids containing the same gRNA operator. The CMV fluoresced twice as bright as the single operator promoter plasmid (denoted by the singular operator site) and about a fourth as bright as the triple operator promoter plasmid (denoted by the triple operator site).
CMV compared to three Gemini single operator promoters. The CMV expressed GFP at a greater intensity than all of the single operator promoters with the sole exception of the promoter containing the g3 operator.
BBa_I712004 compared to four triple operator promoters. The CMV fluoresced anywhere between a third to a fifth as brightly as the four triple operator promoters.
