RNA

Part:BBa_K1614014:Experience

Designed by: Frieda Anna Sorgenfrei   Group: iGEM15_Heidelberg   (2015-09-16)
Revision as of 02:28, 19 September 2015 by Philipp.walch (Talk | contribs)

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Applications of BBa_K1614014

Another biobrick, the ATP Aptamer JAWS1 Spinach2 (BBa_K1614014) was designed in this project as well. Similar to the biobrick BBa_K1614012, we performed in vitro transcriptions to sense ATP in real-time. The construct is a fusion of an ATP aptamer<x-ref>Sassanfar1993</x-ref> and Spinach2<x-ref>Strack2013</x-ref>, which we will call Spinach2-ATP-Aptamer system . To improve the binding of the ATP aptamer to ATP we apply our own implemented JAWS software. Using our software, nucleotides which form the stem region of the ATP aptamer can be predicted, which will improve binding properties of this RNA to ATP advanced the stemming behavior of the ATP Aptamer which was then fused to the Spinach2. Measurements with the spectro fluorometer show that the ATP Aptamer JAWS1 Spinach2 (BBa_K1614014) has a lower fluorescence than ATP Aptamer JAWS2 Spinach2 (BBa_K1614015) which is caused by a weaker steming behavior. Therefore ATP Aptamer JAWS1 Spinach2 is a better candidate for sensing ATP changes during biochemical reactions such as the in vitro transcription. For the in vitro transcription assay the RNA was renatured in 1x Renaturing buffer at 95 °C. 500 nM of the RNA was used for the in vitro transcription for measuring the ATP consumption during transcription. Experiments have shown that the detection range of this ATP sensor which correlates to the transcribed RNA is much more sensitive than traditional techniques that require UV-shadowing. The real time fluorescent readout system even allows the study of enzyme kinetics that depends on ATP.

Figure 1'. Sensing of ATP using the ATP Aptamer Spinach in real time during in vitro transcription.'(A) Assay design of the ATP-Aptamer Spinach2: ATP-AptamerJAWS1 Spinach2 RNA will be applied to a classical in vitro transcription. In presence of ATP, fluorescence emission can be determined. (B) As proof of principle, transcriptions were performed with different concentrations of T7 RNA polymerase. ATP consumption was monitored in real time by measuring the fluorescence of the ATP-AptamerJAWS1 Spinach RNA in regular intervals.(C) To confirm the results of the fluorescence measurements, the in vitro transcription reaction was analyzed using denaturing acrylamide gels.

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