Part:BBa_K4586024
CCP1-Synthetic Notch Receptor
Description and Usage
This composite part codes for a synthetic receptor sensitive to the autoreactive B-cell receptor secreting ACPAs. CCP1 syn notch receptor consists of three main domains, first the extracellular domain represented in tagged citrullinated vimentin conjugated to CD8 alpha signal that mediates the expression of the receptor on the cell membrane, second the transmembrane domain represented in mouse notch core protein that is characterized by mechanosensitive structure that transfer the signal from the extracellular domain to the third intracellular domain that contains our potent transcription module VP64 which trigger the expression of our therapeutic agent (Cas12k\gBAFF-R) as shown in figure 1. and figure 2.
Figure 1 . illustrates the structure of our vimentin synthetic notch receptor, which is formed of 3 components: first, the tagged CCP1 antigen within the extracellular surface of MSCs; second, the mouse notch core protein; and finally, ZF21.16-VP64, representing the internal domain releasing our transcription module VP64
Figure 2. illustrates the design of our genetic circuit coding for the vimentin synthetic notch receptor.
literature characterization of ccp1
The study used gel electrophoresis to structurally characterize CCP1 compared to other parts. In Addition to studying its presence and association with neural tissues in different cross sections.
A)They observed ccp1 in proteins of brain, spinal cord and peripheral nerves.They confirmed that by detection of extra band in HEK293 transfected with ccp1-YFP fusion proteins and absence of signals in pcd mouse brain B)They found relation of ccp1 with myelinated axons(NF200) but not compact myelin (MBP) C)They found ccp1 in motor neurons( CHAT) in the ventral horn of spinal cord .
characterization by mathematical modeling of ccp1
This model is to simulate kinetics of the binding between CCP1 of the Syn-Notch on the surface of the stem cell to BCR of autoreactive B-cell. When stem cells are injected into the body, the amount of free CCP1 of the synthetic notch receptor increases, and as BCR binds to it, the amount of free CCP1 portion decreases, forming a binding state of both that activates the internal domain ZF21.16VP64 of the Syn-Notch
A, Graph(1) shows an increase in the free portion of CCP1 of Syn-Notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line).
Graph(2) shows an increase in the free portion of CCP1 of Syn-Notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line). that dissociates after binding as it is not stable.
Literature Characterization of vp64
vp64 were transfected into HEK293T reporter cells and treated with abscisic acid or DMSO for 48 h.
Statistical significance was calculated with one-way ANOVA, using Dunnett’s multiple testing correction which showed that GFP expression was higher with VP64 than with any other promoter.
charactrization by mathematical modeling of vp64
This internal domain is activated after cell to cell interaction between MSC and B-cell receptor. That leads to increase of transcription factor VP64 that triggers the expression of the internal circuit that secretes the exosome's cargo then cargo loading to exosomes through the loading system (CD63-L7Ae).
As the expression of the internal domain increases (represented as red line), the transcription factor VP64 increases, increasing the expression of the internal circuit for the exosome's cargo to finally produce modified exosomes (represented in blue line).
Experimental Characterization of vp64
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and anti-CD19 and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure in lane (P1)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 774
Illegal NotI site found at 783 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 614
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1399
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