Difference between revisions of "Part:BBa K2100010:Experience"
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===Applications of BBa_K2100010=== | ===Applications of BBa_K2100010=== | ||
| + | We used our promoter to build the [[Part:BBa_K2100031|pPRE4:eYFP]] construct to characterize the functionality of our promoter. | ||
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| + | First, we characterized the synthetic PRE4 promoter in two cell lines: MCF7 and tHESC. All cell lines have endogeneous progesterone Receptor A. We analyzed data from cells induced with induced with 1 uM of MPA compared to those uninduced with hormones. This concentration of MPA was recommended to us as an appropriate "on" or saturating concentration by the Griffith Lab which provided us with these cells. | ||
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| + | We transfected tHESC cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pPRE4:eYFP to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 1 uM MPA. This ratio was chosen to be 1:1 based on the small amount of plasmids being transfected. We were looking to deterimne the on-off functionality of our promoter pPRE4 in the tHESC cell line. | ||
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We characterized our hybrid promoter in both MCF-7 and tHESC cells. The following characterization experiment in tHESC cells tested how the hybrid promoter responds to a 1 uM induction of progesterone (MPA). The cells were transfected with 250 ng of hEF1a-mKate as a transfection marker and 250 ng of pHybrid-eYFP for characterization. | We characterized our hybrid promoter in both MCF-7 and tHESC cells. The following characterization experiment in tHESC cells tested how the hybrid promoter responds to a 1 uM induction of progesterone (MPA). The cells were transfected with 250 ng of hEF1a-mKate as a transfection marker and 250 ng of pHybrid-eYFP for characterization. | ||
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The results showed a 12 fold difference between the yellow fluorescent output of the uninduced MCF-7 cells and the ones induced with 10 nM E2. | The results showed a 12 fold difference between the yellow fluorescent output of the uninduced MCF-7 cells and the ones induced with 10 nM E2. | ||
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===User Reviews=== | ===User Reviews=== | ||
Revision as of 13:11, 21 October 2016
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Applications of BBa_K2100010
We used our promoter to build the pPRE4:eYFP construct to characterize the functionality of our promoter.
First, we characterized the synthetic PRE4 promoter in two cell lines: MCF7 and tHESC. All cell lines have endogeneous progesterone Receptor A. We analyzed data from cells induced with induced with 1 uM of MPA compared to those uninduced with hormones. This concentration of MPA was recommended to us as an appropriate "on" or saturating concentration by the Griffith Lab which provided us with these cells.
We transfected tHESC cells with 250ng of hEF1a:mKate as a transfection marker and 250 ng pPRE4:eYFP to examine the promoter's transcriptional activity by observing increases in yellow fluorescence upon cells being induced with 1 uM MPA. This ratio was chosen to be 1:1 based on the small amount of plasmids being transfected. We were looking to deterimne the on-off functionality of our promoter pPRE4 in the tHESC cell line.
We characterized our hybrid promoter in both MCF-7 and tHESC cells. The following characterization experiment in tHESC cells tested how the hybrid promoter responds to a 1 uM induction of progesterone (MPA). The cells were transfected with 250 ng of hEF1a-mKate as a transfection marker and 250 ng of pHybrid-eYFP for characterization.
The results showed an 100 fold difference between the yellow fluorescent output of the uninduced MCF-7 cells and the ones induced with 10 nM E2.
The next characterization experiment tested how the hybrid promoter responds to a 10 nM induction of E2 (estradiol) in MCF-7 cells. The cells were transfected with 250 ng of hEF1a-mKate as a transfection marker and 250 ng of pHybrid-eYFP for characterization.
The results showed a 12 fold difference between the yellow fluorescent output of the uninduced MCF-7 cells and the ones induced with 10 nM E2.
User Reviews
UNIQf14df38a4984208a-partinfo-00000000-QINU UNIQf14df38a4984208a-partinfo-00000001-QINU