Part:BBa_K2100050
pENTR L4_EGSH 2x k-turn_R1
This basic part entry vector is flanked by L4 and R1 sites, which are used to denote a promoter. This can be cascade with a gene (flanked by L1, L2 sites) using an LR reaction and cloned into a backbone that has a negative selection marker between R4 and R2 sites. This part adheres to RFC 65 for recombination based cloning of mammalian parts.
k-turn stands for kink turn motif in the messenger RNA. The k-turn motif is a part of the L7Ae/k-turn RNA-based repressing system. By putting the k-turn motifs in pENTR promoter vector, the k-turns will be added into the 5'UTR of the gene after LR reaction. The k-turn adds in a higher gene regulation level to reduce the basal expression of the gene downstream due to leaky expression of the EGSH promoter
Characterization Of Kink Turns by AFCM-Egypt 2021
Structural Characterization
Team:AFCM-Egypt 2021has worked on structural characterization of kink turn and in order to model the structural stability of kink-turns riboswitch, we used RNAfold tool that predicts secondary structures of single stranded RNA.
The final result was that the free energy of the thermodynamic ensemble is -15.13 kcal/mol. The frequency of the MFE structure in the ensemble is 25.99 %. The ensemble diversity is 4.35.
Characterization by Mathematical Modelling
In order to simulate the dynamics of the used riboswitches which acts as an essential safety switches in our vaccine design, Mathematical modeling is performed using ordinary differential equations (ODEs) and fitted parameters.
As shown in Figure(2), P2 which represents the binding state shown inhibition throughout the time which conclude inhibition of the circuit. On the other hand, P1 which represents removing the inhibitory effect of riboswitches showed increased transcription of the circuit which reach the steady state after about 200 seconds.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1287
Illegal XbaI site found at 2193
Illegal PstI site found at 1780
Illegal PstI site found at 2200 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1287
Illegal NheI site found at 832
Illegal NheI site found at 1098
Illegal PstI site found at 1780
Illegal PstI site found at 2200
Illegal NotI site found at 2207 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1287
Illegal BglII site found at 1410 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1287
Illegal XbaI site found at 2193
Illegal PstI site found at 1780
Illegal PstI site found at 2200 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1287
Illegal XbaI site found at 2193
Illegal PstI site found at 1780
Illegal PstI site found at 2200 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 709
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