ATP Aptamer JAWS1 Spinach 2.1
This part is an improvement of BBa_K1330000, Spinach2.1. We designed it to be ATP-dependent by joining an ATP aptamer to stem 2 using our software JAWS (http://2015.igem.org/Team:Heidelberg/software/jaws). This part was developed as a tool to sense ATP consumption within biochemical reactions. This part is cloned into the RFC 110 and can be transcribed using the T7 RNA Polymerase directly from Plasmid for further information refer to (File:BBF RFC 110.pdf).
Usage and Biology
The Spinach RNA aptamer binds a small molecule that resembles the fluorophore of the green fluorescent protein (GFP)(Jaffrey, 2011). Upon binding of the small molecule to the Spinach a green fluorescence comparable to that of the GFP is emitted. In our project we use variants of these fluorescent aptamer and engineer them to be dependent on small molecules of our interest.
The ATP aptamer (Sassanfar, 1993) has been previously published. We used both parts together to obtain an ATP-dependent Spinach. To have a RFC 10 compatible version we used the Spinach2.1 as it was described by DTU Denmark in BBa_K1330000.
We measured the fluorescence of the ATP Aptamer JAWS1 Spinach 2.1 (BBa_K1614014) using a spectro fluorometer. Yet, we were able to determine a peak at 500 nm in presence of ATP (Fig 1). Therefore we made the Spinach2.1 (BBa_K1330000) ligand dependent to ATP and even improved our BioBrick BBa_K1614014 by having a higher peak.
Here we imply that the BBa_K1614019 has similar proporties as BBa_K1614014 as listed below.
Proporties of the ATP Aptamer JAWS1 Spinach2
We tested the ATP-dependency of ATP Aptamer JAWS1 Spinach2 by tracking the in vitro transcription of RNA in real-time. During the reaction, the ATP concentration get less and thus the fluorescence we detect reduces over time. The construct is a fusion of an ATP aptamer (Sassanfar 1993) and Spinach2 (Strack 2013), 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 (Fig. 1). 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 has a lower fluorescence than ATP Aptamer JAWS2 Spinach2 (Fig.2) which is caused by a weaker stemming 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. The construct was excited at 460 nm. Emission was measured at 500 nm. 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 (Fig.3).
This part was generated by fusion an ATP aptamer to the Spinach Aptamer (BBa_K1330000). This leads to an ATP-dependent fluorescence. The fluorescence of Spiach2 and Spinach2.1 generated by DTU Denmark show similar fluorescence characteristics. Thus we characterized the ATP-dependency in BBa_K1614014. Here we provide a RFC compatible Spinach2.1 that is ATP-dependent.
1.Sassanfar, M. and J.W. Szostak, An Rna Motif That Binds Atp. Nature, 1993. 364(6437): p. 550-553. 2.Strack, R.L. and S.R. Jaffrey, Live-cell imaging of mammalian RNAs with Spinach2. Methods Enzymol, 2015. 550: p. 129-46. 3. Strack, R.L., M.D. Disney, and S.R. Jaffrey, A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat Methods, 2013. 10(12): p. 1219-24.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 47
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC