Difference between revisions of "Part:BBa K2100008:Experience"
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===Applications of BBa_K2100008===
 
===Applications of BBa_K2100008===
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This entry vector was recombined into an expression vector pPRE3:eYFP with an LR reaction. The construct was characterized in tHESC cells.
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We used our promoter to build the [[Part:BBa_K2100030|pPRE3:eYFP]] construct to characterize the functionality of our promoter.
  
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We transfection pPRE3:eYFP into tHESC to test its on/off functionality by comparing cells induced with MPA and cells induced with 1 uM MPA. We transfected hEF1a:mKate into tHESC as well as a transfection marker.  
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First, we characterized the synthetic PRE3 promoter in two cell lines: ISH and tHESC. All cell lines have endogeneous Estrogen Receptor alpha. 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 pPRE3: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 pPRE3 in the tHESC cell line.
  
 
https://static.igem.org/mediawiki/2016/e/e1/T--MIT--PRE3.png
 
https://static.igem.org/mediawiki/2016/e/e1/T--MIT--PRE3.png

Revision as of 12:52, 21 October 2016


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Applications of BBa_K2100008

We used our promoter to build the pPRE3:eYFP construct to characterize the functionality of our promoter.

First, we characterized the synthetic PRE3 promoter in two cell lines: ISH and tHESC. All cell lines have endogeneous Estrogen Receptor alpha. 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 pPRE3: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 pPRE3 in the tHESC cell line.

T--MIT--PRE3.png

The results showed little to no increase in fold difference of yellow fluorescence when comparing induced vs. uninduced cells. Unfortunately, we could not prove with this experiment that the PRE3 promoter increases activity in response to progesterone induction.

Note: We attempted to characterize pPRE3 in MCF-7 cells as well, however due to low transfection efficiency we were unable to get conclusive results about the on-off functionality of the promoter.

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