Part:BBa_K2429145:Experience

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Verifying the construct

Reporter 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 DF construct. We expected that in absence of our system, there would be an increase yellow fluorescence across the reporter concentrations, as YFP would be translated. In this case, blue fluorescence will be low, because the ribosome will fall off at the stop codon of YFP, before reaching BFP. If our system is present, we expect that levels of blue output will rise with the increase in reporter construct, because YFP will be skipped over, allowing BFP to be produced. These expected results are summarized below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of 3-exon DF reporter 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 DF reporter

3-exon DF 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 show no blue fluorescence in the control condition, but it in fact shows the same amount of yellow fluorescence as blue fluorescence in all conditions. Additionally,adding dCas13a and Ms2 have no effect on the color output.

Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was producing equal levels of blue and yellow in the control condition. '

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.

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