RNA

Part:BBa_K3752002

Designed by: Tudor Onose   Group: iGEM21_Warwick   (2021-10-20)
Revision as of 23:02, 21 October 2021 by PaintMeTed (Talk | contribs)


OXA-48 Long cgRNA

Derived from the parent cgRNA (BBa_K3752000), this cgRNA's sensing domain is longer than the ideal sequence. The longer sequence is theoretically more resistant to mutation, but it is also less specific. Used in screening cgRNA sequences.

Everything below is obtained through computational modelling - no wet lab verification of these data was possible due to time constraints.
All secondary structure simulations were obtained through NUPACK. The data in all the graphs are obtained from a COPASI model, the parameters for which can be found at the bottom of our Engineering page (https://2021.igem.org/Team:Warwick/Engineering).

Below is a NUPACK simulation of the folding of the optimal cgRNA (centre) as opposed to a cgRNA with a shorter sensing loop (left) and this cgRNA (right). All sensing loops labelled in yellow.

T--Warwick--SML.png

The cgRNA with the shorter sensing loop fails to fold into the correct secondary structure at all, making it unable to function at all. The cgRNA with the longer loop folds correctly but the longer sensing domain reduces specificity, making cross-activation more likely.

T--Warwick--SMLG.png

Above is a simulation of the fluorescence obtained through the use of these cgRNAs; as expected, the shorter sensing loop causes no increase in fluorescence with respect to the negative control, whereas both the optimum length and long cgRNAs offer a marked increase in the expression of the fluorescent reporter.

T--Warwick--FlexiblevsDoubleCenter.png

The NUPACK simulation above details the similarities between the cgRNA with the longer sensing loop and a cgRNA with a double centre mismatch. The overall similarity between secondary structures reveals that the longer cgRNA can suffer two mutations in the centre of the sensing loop without completely abolishing function. This is verified in the results of the simulation presented below:

T--Warwick-FlexiblevsDoubleCentreG.png
T--Warwick--OpposingEndsMismatch.png

The secondary structures simulated above do not match the optimum sensing loop cgRNA or the long sensing loop gRNA - they are not functional, which is verified by the simulated results below:

600px

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
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None