Difference between revisions of "Team:BostonU/Description"

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As part of our characterization, this part was also directly compared to several of our Gemini library plasmids, more specifically: <a href = "http://parts.igem.org/Part:BBa_K1875014" style = "color:blue;">BBa_K1875014</a>, <a href = "http://parts.igem.org/Part:BBa_K1875015" style = "color:blue;">BBa_K1875015</a>, <a href = "http://parts.igem.org/Part:BBa_K1875016" style = "color:blue;">BBa_K1875016</a>. 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.</p>
 
As part of our characterization, this part was also directly compared to several of our Gemini library plasmids, more specifically: <a href = "http://parts.igem.org/Part:BBa_K1875014" style = "color:blue;">BBa_K1875014</a>, <a href = "http://parts.igem.org/Part:BBa_K1875015" style = "color:blue;">BBa_K1875015</a>, <a href = "http://parts.igem.org/Part:BBa_K1875016" style = "color:blue;">BBa_K1875016</a>. 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.</p>
  
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<p style = "font-size:150%; padding:5px 150px 15px 150px; color:#0071A7;"> 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.</p>
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As part of our characterization, this part was also directly compared to parts <a href = "http://parts.igem.org/Part:BBa_K1875016" style = "color:blue;">BBa_K1875016</a>, and <a href = "http://parts.igem.org/Part:BBa_K1875018" style = "color:blue;">BBa_K1875018</a> 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.</p>
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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.</p>
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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>
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<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>
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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>
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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>
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Revision as of 05:54, 16 October 2016

Registry Part Validation


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.