Difference between revisions of "Part:BBa K779311"
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| − | <i> Key: Red: mKate, Blue: mKate-Hammerhead, Green: Hammerhead-mKate. | + | <i>The circuit above shows Hef1A constitutive promoter driving an mKate (red fluorescent protein), which serves as our positive control. We also tested Hef1A constitutive promoter driving a hammerhead before an mKate. Once the hammerhead is transcribed, one could assume that since it is self cleaving, the mRNA will be cleaved and the mKate will not be expressed. We also appended the Hammerhead at the 3' end of the mKate mRNA and expect similar results. </i> |
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| + | <i> Above image: Key: Red: mKate, Blue: mKate-Hammerhead, Green: Hammerhead-mKate. | ||
100,000 HEK293 cells were transfected with equimolar amounts of Hef1a:TagBFP, as a transfection marker, and one of the following: Hef1a:mKate, Hef1a:Hammmerhead-mKate, and Hef1a:mKate-Hammerhead. 48 hrs later, cells were harvested and analyzed by flow cytometry until 10,000 events were recorded. The histograms are gated on the blue population of cells, meaning that we are examining red fluorescence (mKate signal) in cells that also received the TagBFP DNA. There is less red fluorescence in the hammerhead constructs compared to the Hef1a: mKate. This suggests that the hammerhead ribozymes could be cleaving the mRNA. </i> | 100,000 HEK293 cells were transfected with equimolar amounts of Hef1a:TagBFP, as a transfection marker, and one of the following: Hef1a:mKate, Hef1a:Hammmerhead-mKate, and Hef1a:mKate-Hammerhead. 48 hrs later, cells were harvested and analyzed by flow cytometry until 10,000 events were recorded. The histograms are gated on the blue population of cells, meaning that we are examining red fluorescence (mKate signal) in cells that also received the TagBFP DNA. There is less red fluorescence in the hammerhead constructs compared to the Hef1a: mKate. This suggests that the hammerhead ribozymes could be cleaving the mRNA. </i> | ||
Latest revision as of 03:47, 11 October 2012
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Along with Part:BBa_K779312, Part:BBa_K779313, and Part:BBa_K779314, this part introduces in vivo hammerhead ribozymes to iGEM and the registry. Hammerhead ribozymes are self-cutting strands of catalytic RNA. After a hammerhead ribozyme is transcribed in a cell, it folds into a secondary structure, which causes self-cleavage. Because of their RNA-cleaving function, hammerheads may be used to regulate protein expression and to make other RNA parts. For more information on hammerheads in general, see http://en.wikipedia.org/wiki/Hammerhead_ribozyme .
These four parts consist of a hammerhead ribozyme attached to an mKate florescent protein for reporting purposes. The two non-repressible hammerheads are designed to show that hammerheads can self-cleave in a mammalian environment. After the hammerhead-mKate sequence is transcribed, the mRNA sequence should cleave, preventing the transcription of mKate. Successful cleavage can be verified by comparing fluorescence levels to a constitutive mKate control. The two repressible hammerheads are designed to test an antisense strategy for interfering with hammerhead function. An interfering RNA should be able to bind to the modified repressible hammerheads, preventing the hammerhead secondary structure from forming and leaving the transcript intact. This can be observed in an increase in fluorescence, when compared to the regular hammerheads.
Testing a 5' Hammerhead appended to mKate:
The circuit above shows Hef1A constitutive promoter driving an mKate (red fluorescent protein), which serves as our positive control. We also tested Hef1A constitutive promoter driving a hammerhead before an mKate. Once the hammerhead is transcribed, one could assume that since it is self cleaving, the mRNA will be cleaved and the mKate will not be expressed. We also appended the Hammerhead at the 3' end of the mKate mRNA and expect similar results.
Above image: Key: Red: mKate, Blue: mKate-Hammerhead, Green: Hammerhead-mKate.
100,000 HEK293 cells were transfected with equimolar amounts of Hef1a:TagBFP, as a transfection marker, and one of the following: Hef1a:mKate, Hef1a:Hammmerhead-mKate, and Hef1a:mKate-Hammerhead. 48 hrs later, cells were harvested and analyzed by flow cytometry until 10,000 events were recorded. The histograms are gated on the blue population of cells, meaning that we are examining red fluorescence (mKate signal) in cells that also received the TagBFP DNA. There is less red fluorescence in the hammerhead constructs compared to the Hef1a: mKate. This suggests that the hammerhead ribozymes could be cleaving the mRNA.
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