Part:BBa_K4140026
Regulatory device
Part Description
The composite component encodes a particular gRNA that is regulated by the human U6 promoter. It is used to direct the Cas12g protein to the target RNA. The interaction of L7Ae with the Kink turn loop inhibits this system, limiting the expression of Cas12g.
Usage
We used this composite as our regulatory system to control the expression of our reporter gene in therapeutic and diagnostic circuits based on CRISPR system which is formed of two-component: The first is Cas12g which is a protein that interacts differently from other Cas proteins in taking RNA as a substrate by making post-transcriptional modification to it that hinders its translation The second part is a gRNA which has an affinity to the target RNA sequence needed to be regulated by CRISPR system activity In our system, This gRNA is designed to target the RNA of PAH to repress its translation as shown in figure 1 and figure 2.
Figure 1. (shows the regulatory circuit in our E.coli-based system.)
Figure 2. (shows our therapeutic E.coli-based system.)
Characterization by mathematical modeling
This model is to simulate the kinetics of the riboswitch (L7Ae with kink turns) that is used in our circuit. The designed circuit is to detect if the increasing substance is either phenylalanine or tyrosine via TyrR. So if phenylalanine level is elevated, L7Ae is formed as it is downstream TyrR that forms a complex via binding with its kink-turn on another circuit; that complex inhibits expression of cas12g, so the circuit will be able to express lacZ alpha (beta-galactosidase) in diagnostic circuit or PAH in the therapeutic circuit. If tyrosine level is elevated with a decreased level of phenylalanine, it activates tyrR inhibitory promoter so no L7Ae would be expressed resulting in cas12g expression to control the circuit as shown figure (4) and graph (1).
Figure (4) illustrates the kinetics of all reactions in riboswitch model
Graph (1) illustrates riboswitch kinetics in which Q represents the condition where L7Ae is expressed and bound to its kink-turns ,therefore inhibiting the expression of cas12g. However, M represents no expression of L7Ae in which cas12g would be expressed to control the circuit if the phenylalanine is absent.
Experimental Characterization
This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 4. The running part (ordered from IDT) included Human u6 Promoter - -Kinkturn - CMV Promoter - Cas12g.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 820
Illegal PstI site found at 1294
Illegal PstI site found at 2236 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 820
Illegal PstI site found at 1294
Illegal PstI site found at 2236 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1815
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 820
Illegal PstI site found at 1294
Illegal PstI site found at 2236 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 820
Illegal PstI site found at 1294
Illegal PstI site found at 2236
Illegal NgoMIV site found at 1737 - 1000COMPATIBLE WITH RFC[1000]
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