Difference between revisions of "Part:BBa K3752001"

Latest revision as of 22:43, 21 October 2021

OXA-48 Short cgRNA

Derived from the parent cgRNA (BBa_K3752000), this sensor's handle is too short to be active - used as a control in screening gRNA 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 this cgRNA (left) and one with a longer sensing loop (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.

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 3: / Line 3: @@
 <partinfo>BBa_K3752001 short</partinfo>
-Derived from the parent cgRNA (BBa_K3752000), this sensor is too short to be active - used as a control in screening gRNA sequences.
+Derived from the parent cgRNA (BBa_K3752000), this sensor's handle is too short to be active - used as a control in screening gRNA sequences.
+<b>Everything below is obtained through computational modelling - no wet lab verification of these data was possible due to time constraints.</b><br>
+<b>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).</b>
+Below is a NUPACK simulation of the folding of the optimal cgRNA (centre) as opposed to this cgRNA  (left) and one with a longer sensing loop (right). All sensing loops labelled in yellow.
+[[File:T--Warwick--SML.png|600px|center]]
+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.
+[[File:T--Warwick--SMLG.png|600px|center]]
+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.
 <!-- Add more about the biology of this part here