Part:BBa_K1614015

ATP Aptamer JAWS2 Spinach2

Created from Part:BBa_K1614011 to sense ATP with a fluorescent read out. The ATP Aptamer JAWS2 Spinach2 is cloned into the RFC 110 and can therefore be transcribed by 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. The stem between the Spinach Aptamer and the ATP aptamer was optimized using our software JAWS (http://2015.igem.org/Team:Heidelberg/software/jaws). This device was applied to detect the small molecule ATP during in vitro reactions such as in vitro transcription. This part compared to the the manually joined one (BBa_K1614012) and BBa_K1614014 the first JAWS optimized ATP-dependent Spinach candidate proofed to have better stronger ATP binding characteristics.

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
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 165
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 165
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 77
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 165
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 165
  Illegal NgoMIV site found at 178
  Illegal NgoMIV site found at 207
  
• 1000
  COMPATIBLE WITH RFC[1000]