RNA

Part:BBa_K1614012

Designed by: Frieda Anna Sorgenfrei   Group: iGEM15_Heidelberg   (2015-09-16)
Revision as of 19:38, 20 September 2015 by Frieda (Talk | contribs) (→‎Usage and Biology)

ATP Aptamer Spinach2

Created from Part:BBa_K1614011 to sense ATP with a fluorescent read out. The part was cloned into the RFC 110 achieving the possibility of transcription using the T7 RNA Polymerase.

Usage and Biology

This part contains the Spinach Aptamer (BBa_K1614011)wherein stem 2 was exchange by an ATP aptamer to make the ligand binding of the Spinach and thus the fluorescence dependent on ATP. This device was applied to detect the small molecule ATP during in vitro reactions such as in vitro transcription. This BioBrick is the first fluorescent ATP sensor. 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). Even though the ATP Aptamer Spinach2 was tested during the in vitro transcription reaction, it showed less sensitivity than the ATP Aptamer JAWS1 Spinach2.

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
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 149
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 149
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 69
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 149
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 149
    Illegal NgoMIV site found at 162
    Illegal NgoMIV site found at 191
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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