Other

Part:BBa_K4351020

Designed by: Laura Keffer-Wilkes   Group: iGEM22_Lethbridge_HS   (2022-10-07)


A scramble of the glucose binding aptamer 1 + RNA Mango

An experimental control sequence that should not bind to glucose. Fluorescent output by RNA Mango gives feedback on level of binding by the aptamer.


RNA was unfolded by heating to 95°C and then let cool to room temperature in the presence of 20 µM ligand. The fluorophore TOI was only added to the RNA just prior to recording the fluorescence spectra and was excited at 510 nm. The emission spectra was recorded between 520 - 600 nm and two scans were completed.

resultsfig4.jpg

From our experiments, we concluded that the addition of glucose to the RNA biosensor mix does result in an increase in fluorescence but only under certain conditions (Figure 4A). To use this biosensor to measure glucose binding, it first needed to be unfolded by heating to a high temperature and then allowed to refold and cool in the presence of glucose. The TOI was then added just prior to measuring the fluorescence. Our hypothesis is that the RNA is quite conformationally stable and cannot unfold to bind to glucose without first being melted. TOI can also not be added first, as it has a low nanomolar affinity for RNA Mango and would therefore bias the RNA folding to form the RNA Mango structure regardless of whether glucose was present or not.

Unfortunately, the scramble sequence aptamer also appears to bind to glucose (Figure 4C), as the fluorescence signal increased in the presence of ligand. This indicates that the RNA sequence we chose for the glucose aptamer is not specific enough and needs to be retested.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 89


[edit]
Categories
Parameters
None