RNA

Part:BBa_K1614018

Designed by: Daniel Heid   Group: iGEM15_Heidelberg   (2015-09-18)

ATP Aptamer Spinach 2.1

The ATP Aptamer Spinach2.1 is the first fluorescent ATP sensor that was designed for the in vitro transcription tool box designed by the iGEM Team Heidelberg 2015. This ATP Aptamer Spinach2.1 is cloned into the RFC 110.

Usage and Biology

ATP Aptamer Spinach 2.1 is a fusion of the BBa_K1330000 generated by DTU Denmark. In this part we provide a RFC 10 compatible Spinch2.1 variant that responds on the presence of ATP. This Spinach 2.1 is designed ligand dependent on ATP and should find its use in the detection of the small molecule ATP. Applications such as nucleotide sensing during in vitro transcription are possible.


Proporties of the ATP Aptamer Spinach2

According to the results from DTU Denmark on BBa_K1330000 the Spinach2.1 behaves similar like Spinach2 BBa_K1614011 thus the here presented part should behave like BBa_K1614012. The switching ability of the Spinach depends on the exchanged stem. BBa_K1614012 contains a Spinach2 aptamer that was fused to the ATP Aptamer. This device was applied to detect the small molecule ATP during in vitro reactions such as in vitro transcription. Experiments have shown that the ATP Aptamer Spinach2 is able to detect ATP and can even sense dynamic changes in ATP concentration during biochemical reactions like in vitro transcriptions. Compared to the other ATP Aptamer Spinach2 variations the ATP Aptamer Spinach2 has the lowest fluorescence shown in the spectrum (Fig 1.). 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_K1614012. Here we provide a RFC compatible Spinach2.1 that is ATP-dependent.

Fig. 1.Establishment of a system to sense small molecule using the Spinach2 Aptamer. (A) Emission spectrum of the original Spinach2 Aptamer, which was applied as an internal control. (B) As another internal control, we reproduce the data for the c-di-GMP Spinach2 system, published by Kellenberger et al.. Indeed, highest fluorescence maximum for the c-di-GMP Spinach2 system was measured in presence of the ligand. (C) Analysis of the fluorescent properties of our ATP Aptamer Spinach2 constructs. The Spinach2 containing the Szostak ATP Aptamer shows the lowest fluorescence of all three ATP Aptamer Spinach2 variations. The JAWS-generated ATP AptamerJAWS1 Spinach2 and the ATP AptamerJAWS2 Spinach2 show higher fluorescence maxima in presence of ATP.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 39
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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