Part:BBa_K1442006:Experience

Experimentation

We used a system including 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.

Method

We transfected the cells with 0.1μg of the aptazyme or control RNA, transcribed in vitro using Invitrogen RNA transcription kit, into each well of cells in a 96-well plate. The cells were plated at a concentration of 0.15x10⁵ determined using a haemocytometer. The cells were transfected using lipofectamine delivery in which lipid nanoparticles details of which can be found at Biontex K2 transfection system. Flourescence of the media and cells themselves were also taken into account. Each IRES had 5 biological repeats in each cell type with and without theophylline while the control had 5. Technical repeats were very difficult to achieve as it required splitting of the cells in each well as both HeLa and Huh 7.5 are attached cells, therefore the likelihood of spillage and contamination between the wells was too high to warrant the added accuracy of technical repeats.

We added theophylline to half the wells that contained cells with aptazyme DNA at a concentration of 4mM.

Results

As in the IRES experiment the tecan Magellan is not able to supply CO2 to the cells in the 96 well plate hence they were growth overnight to allow translation of the GFP and then put in the plate reader where the cells gradually died. We therefore used time point which signified the maximal GFP reading for the majority of cells, 1800s, and compared results at this point. The graph below shows how fluorescence changed over time and the general peak is clear.

An interesting note from this is that the theophylline seems to extend the flourescent period of the cells perhaps providing extra nutrients and extending the life span of the cells as in both cell types the peak of flourescence is lower at 1800s seconds but remain much higher than the same cells without theophylline as seen at 80000s.

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.

If time permitted it would also be useful to test the efficacy of the aptazyme with different concentrations of theophylline as the concentration used was reduced from that used in the Klauser and Hartig (2013) paper as theophylline quickly precipitates at room temperature and we were unable to achieve the concentrations required.

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