Difference between revisions of "Part:BBa K2100031:Experience"

 
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===Applications of BBa_K2100031===
 
===Applications of BBa_K2100031===
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pPRE4:eYFP is a cascade of our synthetic promoter [[Part:BBa_K2100009|pPRE4]] with a yellow florescent tag, [[Part:BBa_K2100005|eYFP]]. The parts were combined through a gateway cloning technique called an LR reaction.
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We characterized the cascade of our synthetic promoter PRE3 with the eYFP gene in two cell lines: MCF7 and tHESC. All cell lines have endogeneous progesterone receptor A. We analyzed data from cells 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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https://static.igem.org/mediawiki/2016/e/ed/T--MIT--PRE4tHESC.png
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The panel above represents results for pPRE4:eYFP transfected into the tHESC cell line, comparing the uninduced population to a population induced with 1 uM MPA. The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.
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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 PRE4 promoter increases activity in response to progesterone induction.
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Characterizing the PRE4 promoter in MCF-7 cells showed more promising functionality. We transfected pPRE4:eYFP and hEF1a:mKate as a transfection marker into MCF-7. 250 ng of each construct was used, maintaining a 1:1 ratio of DNA. Like the tHESC experiment, the cells were induced with 1 uM of MPA and compared with an uninduced control.
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https://static.igem.org/mediawiki/parts/5/57/T--MIT--khb_pp4onoffmcf7.jpeg
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The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.
 +
 +
While the results are not definitive due to the poor transfection efficiency, pPRE4 has approximately a five fold increase in activity when induced with MPA for cells with lower amounts of plasmid as seen by the large fold difference at lower levels of red florescence -- our transfection marker. We can believe that with a better transfection efficiency, the cells that do receive a large amount of plasmids will demonstrate a similar fold difference between on and off when induced with MPA.
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Overall, our promoter pPRE4 cascade with eYFP demonstrated limited success of progesterone signaling sensing in MCF7.  This construct did allow us to characterize the exact activity of our synthetic promoter, pERE3, in multiple cell lines by quantifying the yellow florescence expression in comparison to the red florescence expression.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 13:59, 21 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2100031

pPRE4:eYFP is a cascade of our synthetic promoter pPRE4 with a yellow florescent tag, eYFP. The parts were combined through a gateway cloning technique called an LR reaction.

We characterized the cascade of our synthetic promoter PRE3 with the eYFP gene in two cell lines: MCF7 and tHESC. All cell lines have endogeneous progesterone receptor A. We analyzed data from cells 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.

T--MIT--PRE4tHESC.png

The panel above represents results for pPRE4:eYFP transfected into the tHESC cell line, comparing the uninduced population to a population induced with 1 uM MPA. The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.

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 PRE4 promoter increases activity in response to progesterone induction.

Characterizing the PRE4 promoter in MCF-7 cells showed more promising functionality. We transfected pPRE4:eYFP and hEF1a:mKate as a transfection marker into MCF-7. 250 ng of each construct was used, maintaining a 1:1 ratio of DNA. Like the tHESC experiment, the cells were induced with 1 uM of MPA and compared with an uninduced control.

T--MIT--khb_pp4onoffmcf7.jpeg

The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker.

While the results are not definitive due to the poor transfection efficiency, pPRE4 has approximately a five fold increase in activity when induced with MPA for cells with lower amounts of plasmid as seen by the large fold difference at lower levels of red florescence -- our transfection marker. We can believe that with a better transfection efficiency, the cells that do receive a large amount of plasmids will demonstrate a similar fold difference between on and off when induced with MPA.

Overall, our promoter pPRE4 cascade with eYFP demonstrated limited success of progesterone signaling sensing in MCF7. This construct did allow us to characterize the exact activity of our synthetic promoter, pERE3, in multiple cell lines by quantifying the yellow florescence expression in comparison to the red florescence expression.

User Reviews

UNIQ0c4392973725420d-partinfo-00000000-QINU UNIQ0c4392973725420d-partinfo-00000001-QINU