Part:BBa_K2429045:Experience

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Tre:Ms2 DOX Induction

Once we had determined a proper amounts of mKate-ff4 and ASO plasmid, we wanted to check how much of an effect the Ms2 was having on ASOs with hairpin loops (ASO+). We expected that as the amount of Ms2 increased in the system, more Ms2 would be available to bind the hairpin loops, and lead to increased blocking of the spliceosome. After a certain amount of Ms2 was made, the system would become saturated with Ms2 and the knockdown would level-off. The expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we put Ms2 downstream of Tre, which is a DOX inducible promoter. The more DOX we added to the system, the higher Ms2 production we would see off the Tre promoter. We transfected a constant 300 ng of Tre:Ms2, and varied the amount of DOX from 5 to 500 uM. We ran DOX inductions for ASO0+, ASO2+ and ASO4+. (For a detailed explanation of how to plan a mammalian transfection click here)

Tre:Ms2 DOX Induction for ASO0+

DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO0+. The color of the line indicate the transfection bins of each result.

Results for ASO0+ showed that as levels of DOX, and therefore Ms2 levels, rose in the system, there was a decrease in red fluorescence.

Tre:Ms2 DOX Induction for ASO2+

DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO2+. The color of the line indicate the transfection bins of each result.

Results for ASO2+, unexpectedly, show a slight increase in red across DOX concentrations.

Tre:Ms2 DOX Induction for ASO4+

DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO2+. The color of the line indicate the transfection bins of each result.

Results for ASO4+ show a slight decrease in red across DOX concentrations.

The results from ASO0+ show a log decrease in DOX concentrations, indicating that our system is functioning as expected. Based on these results we concluded that maximizing the amount of Ms2 in our system was preferable. Therefore, we decided to transfect with 300 ng of Ms2 moving forward, as this was the maximum amount of DNA per plasmid we transfected.

Based on these results, we decided to transfect with 300 ng of Ms2 moving forward.

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