Difference between revisions of "Part:BBa K779200"
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<b> Testing the Hef1A promoter with eYFP and with mKate: </b>  
 
<b> Testing the Hef1A promoter with eYFP and with mKate: </b>  
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<i> This figure shows HEK293 cells which were transiently transfected with Hef1A:TagBFP and Hef1A:mKate using Lipofectamine 2000. Equimole amounts of DNA were delivered, 500 ng total per 1.65 uL of reagent. Images were taken on a Zeiss microscope at 10X. <strong>(a)</strong> brightfield <strong>(b)</strong> overlay of blue, red and brightfield <strong>(c)</strong> blue channel <strong>(d)</strong> red channel</i>
 
<i> This figure shows HEK293 cells which were transiently transfected with Hef1A:TagBFP and Hef1A:mKate using Lipofectamine 2000. Equimole amounts of DNA were delivered, 500 ng total per 1.65 uL of reagent. Images were taken on a Zeiss microscope at 10X. <strong>(a)</strong> brightfield <strong>(b)</strong> overlay of blue, red and brightfield <strong>(c)</strong> blue channel <strong>(d)</strong> red channel</i>
  
 
<b> Testing the Hef1A promoter with rtTA3 and with TagBFP: </b>
 
<b> Testing the Hef1A promoter with rtTA3 and with TagBFP: </b>
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<i>100,000 HEK293 cells were transfected with equimolar rations of Hef1a:rTTA3, TreT:mKate, and Hef1a:TagBFP (as a transfection marker), standardized to 500 ng total plasmid DNA, with 1.65 uL Lipofectamine 2000.  After 48 hours, cells were imaged on a Leica Confocal Microscope at 10x. We can see that we have transfection, since cells are fluorescing blue. Also, we can see that as we increase the concentration of DOX present, we see an increase in red fluorescence. </i>
 
<i>100,000 HEK293 cells were transfected with equimolar rations of Hef1a:rTTA3, TreT:mKate, and Hef1a:TagBFP (as a transfection marker), standardized to 500 ng total plasmid DNA, with 1.65 uL Lipofectamine 2000.  After 48 hours, cells were imaged on a Leica Confocal Microscope at 10x. We can see that we have transfection, since cells are fluorescing blue. Also, we can see that as we increase the concentration of DOX present, we see an increase in red fluorescence. </i>

Revision as of 21:20, 8 October 2012

Hef1A MammoBlock

Promoter Vector containing human elongation factor 1a (hEF1a) promoter; acting as a low level constitutive promoter.

We have tested this promoter with many different fluorescent proteins (TagBFP, eYFP, mKate) as well as activators. We have used the Gateway method to create composite parts with the Hef1A promoter: Hef1A:eYFP - BBa_K779600, Hef1A:mKate - BBa_K779601, Hef1A:TagBFP-4xFF5 - BBa_K779602, Hef1A:eYFP-4xFF1 - BBa_K779603, Hef1A:rtTA3 - BBa_K779604

Testing the Hef1A promoter with eYFP and with mKate: ConstColorsMIT.png

This figure shows HEK293 cells which were transiently transfected with Hef1A:TagBFP and Hef1A:mKate using Lipofectamine 2000. Equimole amounts of DNA were delivered, 500 ng total per 1.65 uL of reagent. Images were taken on a Zeiss microscope at 10X. (a) brightfield (b) overlay of blue, red and brightfield (c) blue channel (d) red channel

Testing the Hef1A promoter with rtTA3 and with TagBFP: DOXCURVEpics.png
100,000 HEK293 cells were transfected with equimolar rations of Hef1a:rTTA3, TreT:mKate, and Hef1a:TagBFP (as a transfection marker), standardized to 500 ng total plasmid DNA, with 1.65 uL Lipofectamine 2000. After 48 hours, cells were imaged on a Leica Confocal Microscope at 10x. We can see that we have transfection, since cells are fluorescing blue. Also, we can see that as we increase the concentration of DOX present, we see an increase in red fluorescence.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 315
    Illegal PstI site found at 820
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 315
    Illegal PstI site found at 820
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 569
    Illegal XhoI site found at 968
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 315
    Illegal PstI site found at 820
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 315
    Illegal PstI site found at 820
    Illegal NgoMIV site found at 703
    Illegal AgeI site found at 81
  • 1000
    COMPATIBLE WITH RFC[1000]