Part:BBa_K4586022
ZF21.16-VP64
Part Description
This part is coding for our intracellular transcription module, which is formed of 3 elements: the first is zinc finger 21.16, which drives our transcription module to its promoter; the second is nuclear localization signal SV40, which mediates the entrance of the transcription unit into the nucleus; the previous two elements are linked through a G4S linker in between; and the third is VP64, which is a potent transcription factor.
Usage
This part is implemented in our design to represent the internal domain of the syn notch receptor, which controls the level of our therapeutic agent by releasing the transcription factor VP64, which triggers the expression of the internal circuit that secretes the exosomes' cargo, which is regulated by ZF 21-16 minCMV promoter activity as shown in figure 1.
Figure 1: This figure illustrates the construction of ZF21.16-VP64 that represents the internal domain of our synthetic notch receptor expressed on engineered MSC.
Literature Characterization
The study investigate the effect of activation domain (AD) and different promoters on gene expression with selection of promoter (two factor ANOVA p<0.001) and choice of AD(p<0.001)and interaction between variables(p<0.001)
So they found that mutant ZF increased gene expression with VP64 more than VP16(one-tailed Welch’s t-test,P<0.05)and with VPR more than VP64(one-tailed Welch’s t-test,P<0.01). All VPR-ZF caused the same expression in wt and mutant ZF (turkey HSD test with α = 0.05).
charactrization by mathematical modelinge
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
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 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.
References
H. I., Bagheri, N., & Leonard, J. N. (2020). The COMET toolkit for composing customizable genetic programs in mammalian cells. Nature communications, 11(1), 779. https://doi.org/10.1038/s41467-019-14147-5
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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