Difference between revisions of "Part:BBa K3752002"

Revision as of 22:50, 21 October 2021

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.

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.

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.

The NUPACK simulation above

Sequence and Features