RNA

Part:BBa_K1614020

Designed by: Daniel Heid   Group: iGEM15_Heidelberg   (2015-09-18)
Revision as of 14:58, 20 September 2015 by Frieda (Talk | contribs) (→‎Usage and Biology)

ATP Aptamer JAWS2 Spinach 2.1

This tool was designed to detect ATP in live cells and in vitro during biochemical reactions. This useful tool was cloned into the RFC 100 and can be transcribed using the T7 RNA Polymerase.


Usage and Biology

ATP Aptamer JAWS2 Spinach 2.1 is a fusion of the BBa_K1330000 generated by DTU Danemark. According to our results, this fusion should contain similar properties as BBa_K1614014. Yet, this Spinach 2.1 is ligand dependend to ATP and should find its use in the detection of the small molecule ATP. Applications such as nucleotide sensing during in vitro transcription or because of its strong stem live cell imaging are possible.

Proporties of the ATP Aptamer JAWS2 Spinach2

Along with the ATP Aptamer JAWS1 Spinach2 (BBa_K1614014), ATP Aptamer JAWS2 Spinach2 (BBa_K1614015) was generated by the software JAWS that was designed by the iGEM Team Heidelberg 2015. The ATP Aptamer JAWS2 Spinach2 was transcribed using T7 RNA Polymerase and purified by denaturing polyacrylamide gel electrophoresis. Fluorescence was achieved only in presence of ATP and DFHBI if excited at 460 nm. Spectra have shown that the ATP Aptamer JAWS2 Spinach2 has the highest peak of all tested ATP Aptamer Spinach2 variations (Figure 1). This ATP dependent Spinach2 was designed for the detection of ATP in dynamic biochemical reactions, but its strong stemming does not allow the sensing of ATP in dynamic processes. Hence, sensor can be probably applied for in vivo imaging experiments.

Figure 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 47
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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