Part:BBa_K1614020

ATP Aptamer JAWS2 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

ATP Aptamer JAWS2 Spinach 2.1 is a fusion of the BBa_K1330000 generated by DTU Denmark. According to our results, this fusion should contain similar properties as BBa_K1614015. This Spinach 2.1 is 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 or because of its strong stem it can be used for live cell imaging.

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. 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_K1614015. Here we provide a RFC 10 compatible Spinach2.1 that is ATP-dependent.

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]