This part is a fusion of Spinach2.1 desinged by the iGEM Team DTU Danemark. By cloning the part into the RFC 110 transcription is possible using T7 RNA Polymerase. Cleavage is achieved by the HDV.
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Cloned without restriction enzymes to avoid issues occurring from conserved cut sites. For more details see RFC 110.
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The ATP Aptamer Spinach2 is generated by fusion of Spinach2 and an ATP Aptamer which exchanged the second stem loop of the Spinach2 construct. By exchanging the loop we achieved a ligand depencency to ATP and therefore achieved just a fluorescence in prencence of DFHBI and ATP.
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==Proporties of the ATP Aptamer Spinach2 ==
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In our project we generated new biobricks. The first one (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. 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.
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Even though the ATP Aptamer Spinach2 was tested during the in vitro transcription reaction, it showed less sensitivity than the ATP Aptamer JAWS1 Spinach2.
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[[File:Spectra.png|200 px|left|Establishment of a system to sense small molecule using the Spinach2 Aptamer. (A)</b> Emission spectrum of the original Spinach2 Aptamer, which was applied as an internal control. <b>(B)</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. <b>(C)</b> 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.]]
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===Source===
===Source===
Revision as of 14:45, 20 September 2015
ATP Aptamer Spinach 2.1
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]
Design Notes
Cloned without restriction enzymes to avoid issues occurring from conserved cut sites. For more details see RFC 110.
The ATP Aptamer Spinach2 is generated by fusion of Spinach2 and an ATP Aptamer which exchanged the second stem loop of the Spinach2 construct. By exchanging the loop we achieved a ligand depencency to ATP and therefore achieved just a fluorescence in prencence of DFHBI and ATP.
