Difference between revisions of "Part:BBa K4367012"
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<partinfo>BBa_K4367012 short</partinfo>
 
<partinfo>BBa_K4367012 short</partinfo>
  
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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. <br>
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The function of dCas9-nTEV, together with dCas9-cTEV and gRNA, is as a modular DNA sensor that responds by increasing the enzymatic activity of the split TEV if the target DNA is present. <br><br>
  
 
<h2>Description</h2>
 
<h2>Description</h2>
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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 [https://www.nature.com/articles/s41556-021-00836-1]. 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. <br><br>
+
This protein is a fusion of dCas9 and nTEV with a GS-linker (nTEV: BBa_K4367010). With gRNA, it will be directed towards binding to a specific sequence of DNA, without cutting it like Cas9 would do, but it would just bind to the DNA. 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. <br><br>
  
 
<strong>Figure 1. The dCas9-splitTEV system binding to DNA and inducing complementation of split TEV.</strong> <br>
 
<strong>Figure 1. The dCas9-splitTEV system binding to DNA and inducing complementation of split TEV.</strong> <br>
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<h2>Future design considerations</h2>  
 
<h2>Future design considerations</h2>  
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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.
+
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. <br><br>
 +
 
 +
It was elaborated in the part TEVp (BBa_K4367010) that there exists multiple possible proteases that have specific recognition sites and can be split like TEVp. The following proteins was simulated in AlphaFold with promising results: dCas9-nPPV, dCas9-nSbMV, dCas9-nSuMMV, dCas9-nHRV_3C, dCas9-cHRV_3C. The simulations are shown in figure 3 and figure 4. These simulations displayed that the folding of dCas9 was correct and that the split protease was essentially free floating outside of dCas9 but still anchored to it with a linker.<br><br>
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<strong>Figure 3. Simulation in AlphaFold on the fusion between dCas9 and the following proteases: nPPVp (yellow), nSbMV (red) and nSuMMV (blue).</strong> <br>
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[[File:Alternative dCas9-splitProteases.png|600px]] <br><br>
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<strong>Figure 4. A simulation in AlphaFold where nHRV_3C (red) and cHRV_3C (cyan) are bound to dCas9.</strong> <br>
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[[File:AlphaFold dcas9-splitHRV3C.png|600px]] <br><br>
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A simulation on nTEV-dCas9 and cTEV-dCas9 (opposite order as in the parts) showed worrying results that the split TEV may distrub the folding of dCas9 by binding to the insides of dCas9. It is unclear how representative this result is for dCas9-nTEV and dCas9-cTEV (observe the order!), but it would be a good idea to simulate these in AlphaFold if one were to use this part. If the results are still worrying, it would reasonable to examine the alternative proteases.<br><br>
  
 
<h2>Sequence and Features</h2>  
 
<h2>Sequence and Features</h2>  

Revision as of 19:18, 11 October 2022


dCas9-nTEV

The function of dCas9-nTEV, together with dCas9-cTEV and gRNA, is 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, it will be directed towards binding to a specific sequence of DNA, without cutting it like Cas9 would do, but it would just bind to the DNA. 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.
Schematic dCas9-splitTEV.png

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.
System dCas9-splitTEV and iGal.png


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.

It was elaborated in the part TEVp (BBa_K4367010) that there exists multiple possible proteases that have specific recognition sites and can be split like TEVp. The following proteins was simulated in AlphaFold with promising results: dCas9-nPPV, dCas9-nSbMV, dCas9-nSuMMV, dCas9-nHRV_3C, dCas9-cHRV_3C. The simulations are shown in figure 3 and figure 4. These simulations displayed that the folding of dCas9 was correct and that the split protease was essentially free floating outside of dCas9 but still anchored to it with a linker.

Figure 3. Simulation in AlphaFold on the fusion between dCas9 and the following proteases: nPPVp (yellow), nSbMV (red) and nSuMMV (blue).
Alternative dCas9-splitProteases.png

Figure 4. A simulation in AlphaFold where nHRV_3C (red) and cHRV_3C (cyan) are bound to dCas9.
AlphaFold dcas9-splitHRV3C.png

A simulation on nTEV-dCas9 and cTEV-dCas9 (opposite order as in the parts) showed worrying results that the split TEV may distrub the folding of dCas9 by binding to the insides of dCas9. It is unclear how representative this result is for dCas9-nTEV and dCas9-cTEV (observe the order!), but it would be a good idea to simulate these in AlphaFold if one were to use this part. If the results are still worrying, it would reasonable to examine the alternative proteases.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4636
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 14
    Illegal BsaI.rc site found at 4643