Part:BBa_K2429051:Experience
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Applications of BBa_K2429051
Verifying the construct
mKate Titration
Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the 3-exon mKate-HBG construct. We expected that in absence of our system, there would be no mKate fluorescence, as the stop codon in the REST exon will be included. If our system is present, we expect that levels of red output will rise with increase in reporter construct. These expected results can be seen below. The different colored lines corrispond to different transfection bins
In our experiment, we varied the amounts of 3-exon mKate-HBG from 10 to 500 ng. We also transfected Guide3 with dCas13a, and ASO2+ with Ms2 at optimized amounts from earlier experiments. (For a detailed explanation of how to plan a mammalian transfection click here)
mKate Titration for 3-exon mKate-HBG
3-exon mKate-HBG reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bins of each result.
We expected that the reporter would produce no fluorescence, but our results here show normal output levels for mKate across the reporter concentrations. Additionally, as shown below adding dCas13a and Ms2 have no effect on the red output.
3-exon mKate-HBG titration with ASO2+ 3-exon mKate-HBG titration with Guide3
Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.