Difference between revisions of "Part:BBa K3752002"

Latest revision as of 23:08, 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 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.

The secondary structures simulated above do not match the optimum sensing loop cgRNA or the long sensing loop gRNA - they would be inactive.

Both of the assumptions are verified through the results of the simulation represented in the graph below. The cgRNA with two mutations in its long sensing loop still presents some activity whereas the ones with mutations at the ends present no activatory activity. The longer sensing loop hence offers limited resistance to mutation but presents less specificity.

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]

@@ Line 20: / Line 20: @@
 [[File:T--Warwick--FlexiblevsDoubleCenter.png|600px|center]]
-The NUPACK simulation above
+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.
+[[File:T--Warwick--OpposingEndsMismatch.png|600px|center]]
+The secondary structures simulated above do not match the optimum sensing loop cgRNA or the long sensing loop gRNA - they would be inactive.
+Both of the assumptions are verified through the results of the simulation represented in the graph below. The cgRNA with two mutations in its long sensing loop still presents some activity whereas the ones with mutations at the ends present no activatory activity. The longer sensing loop hence offers limited resistance to mutation but presents less specificity.
+[[File:T--Warwick-FlexiblevsDoubleCentreG.png|600px|center]]
 <!-- Add more about the biology of this part here