Part:BBa_K1442006:Experience

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Experimentation

We used a system including an EMCV IRES, a human optimised GFP coding sequence and the aptazyme in that order to test the self-splicing efficiency and effect of theophylline on the aptazyme. We initially needed to determine whether the theophylline concentration we used, 4mM, affects growth or fluorescence independent of the aptazyme.

This bar chart demonstrates the effect of theophylline on HeLa and Huh cells without an aptazyme present.

We added theophylline at a concentration of 4mM to the EMCV IRES and compared this with the EMCV IRES in the cells without theophylline. As seen in the bar chart above there was no significant difference in fluorescence in HeLa cells whereas in Huh cells there was an increase when theophylline was added, in this case there were only three data points for the EMCV testing with theophylline and would need further investigation to see if this significance was a type II error or not.

We tested the aptazymes efficiency in vivo in two cell types; HeLa and Huh cells seperately and compared the fluorescence when theophylline was absent and present in a concentration of 4mM. The RNA strand was transfected into the cells using a lipofectamine protocol which forms lipid nanoparticles containing the mRNA which is then transported into cells. The self-splicing occurring following the conformational change induced by theophylline binding destabilises the RNA strand and induces degradation of the entire strand therefore reducing fluorescence.

Aptazyme theophylline induced self-splicing ability demonstrated in HeLa and Huh 7.5 cells.

This bar chart indicates that there is a significant reduction in fluorescence in HeLa cell expressing an RNA strand encoding GFP and a regulatory aptazyme element. There is a 22% reduction in those HeLa cells with theophylline added than when the RNA strand remains intact. In Huh7.5 cells, however, this requires more testing to be seen as significant or non-significant as the error bars overlap but looks to be moving towards significant if more data points were to be investigated.

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