Part:BBa_K4367012
dCas9-nTEV
The function of dCas9-nTEV, together with dCas9-cTEV and gRNA, is to act as a modular DNA sensor that responds by increasing the enzymatic activity of the split TEV if the target DNA is present.
Description
This protein is a fusion of dCas9 and nTEV with a GS-linker (nTEV: BBa_K4367010). With gRNA, dCas9 will just bind to the target DNA without cutting it like Cas9 would do [1]. With appropriate gRNA, dCas9-nTEV and its counterpart dCas9-cTEV, they would bind close to each other if a specific DNA sequence is present. This would bring nTEV and cTEV much closer to each other on average than if there was no target DNA present, which would cause a greater amount of nTEV and cTEV complementing each other to regain its enzymatic function (). In conclusion, the presence of a specific DNA sequence would induce a higher enzymatic activity of the split TEV. This is illustrated in figure 1.
Figure 1. The dCas9-splitTEV system binding to DNA and inducing complementation of split TEV.
Usage
The dCas9-splitTEV system is used as a DNA sensor which will activate a reporter protein that responds to the enzymatic activity of TEV. One reporter is the FRET Protein (FP: BBa_K4367011), which is a pair of fluorescent proteins bound together by a cleavable linker. If the linker is cleaved by TEV, the FRET phenomnea will disappear, which would be measurable with appropriate lab equipment. The other reporter protien is Inhibited Beta-galactosidase (iGal: BBa_K4367009), which will undergo Alpha-complementation when cleaved by TEV and produce a blue pigment visible by eye.
The dCas9-splitTEV system alongside iGal is illustrated in figure 2.
Figure 2. The Cell-free Modular DNA Detection Device. dCas9-splitTEV binds to DNA and activates iGal to produce a visible readout.
Future design considerations
The GS-linker in the part is 20 amino acids long, although that may or may not be optimal depending on the distance between where the pair of dCas9 binds. A longer distance reasonably would result in a longer optimal linker, but that relationship would have to be established experimentally. Our software, PLOP, uses a model to predict the likelyhood of the halves of the split TEV being in each others proximity, which could gives a clue to what types of gRNA and length of linkers would be reasonable to test. We intended to test multiple linkers and gRNA to test our model and find an optimal combination of these, but that turned out to require more time than our timeframe - The potential of this system has not yet been explored.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4636
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 14
Illegal BsaI.rc site found at 4643
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