Difference between revisions of "Part:BBa K1442040"
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<partinfo>BBa_K1442040 short</partinfo>
 
<partinfo>BBa_K1442040 short</partinfo>
  
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The MS2 bacteriophage coat protein is a translational repressor [4] that binds the MS2 hairpin to encapsidate the genome and represses replicase synthase translation [1]. Exploiting the MS2 box-protein binding relationship is known as MS2 tagging.
 
  
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Isaacs et al [2] noted that the MS2 protein binding box is of use for engineering RNA transcriptional activators via the DNA-protein binding interactions and the hairpin has thus been shown that the MS2 box can be used to drive expression of proteins when localised to a promoter (e.g. the MS2 protein is fused to a DNA-binding protein and the MS2 box is localised to its target promoter, driving expression).
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Regulating the expression of a protein can be achieved through repressive elements; we have the MS2 and MS2 hairpin, which regulate the production of RNA Dependent RNA polymerase (RdRp). As RdRp is fused with MS2 through a P2A linker, an indefinite increase in RdRp production is rate-limited by MS2 production. MS2 is able to bind to the MS2 hairpin sequence, also rate-limiting RdRp production, which in turn regulates the whole replicon system. The iGEM community can benefit from a regulation system, in which MS2 can be fused to a protein of interest to regulate its expression. If the fusion protein is non-functional, engineering restriction sites in the P2A linker and site specific ligation reconstitutes RdRp without the MS2 hairpin. The part derives from Auslander et al, 2012, which showed that the MS2 protein-box relationship can be designed for use in synthetic biology to create programmable single-cell mammalian biocomputers with simple expression logic, increasing the complexity of an information processing system without researcher input.
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=='''Characterization & Improvement by <html><a href="https://2021.igem.org/Team:AFCM-Egypt">AFCM-Egypt 2021</a></html>'''==
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===Improvement by addition of a Destabilizing domain===
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Team:<html><a href="https://2021.igem.org/Team:AFCM-Egypt">AFCM-Egypt 2021</a></html>has improved this part As shown in <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K3743004">BBa_K3743004</a></html> by addition of destabilization domains (DD) which shows a fusion protein component that is unstable and destabilizes other proteins, leading to protein degradation. A well-known example of DD is mutated DHFR (E. coli) (DDDHFR), which causes the degradation of proteins in cells. However, if a trimethoprim analog (TMP) is added to bind the destabilizing domain, the fusion protein can stabilize and return the expression to normal levels. This approach allows the regulation of secreted proteins and their biological activity, allowing functional control over proteins of interest in a variety of contexts in mammalian cell cultures, the Plasmodium and Toxoplasma apicomplexans, and in live mice, creating new avenues to open various applications such as cancer therapy and targeted gene delivery. In our part, the destabilizing domain binds to the ms2 coat protein that inhibits it from binding to its riboswitch until trimethoprim analog (TMP) is added which acts as a small molecule inhibitor will free the MS2 after stabilizing DD. Free MS2 inhibits the circuit to be able to control the transcription process in extreme conditions as cytokine storm or unpredictable results. This part represents the final form of the used riboswitch to be controlled by Trimethoprim (TMP) that has a small molecule inhibitor role. Destabilizing domain (DD) is fused to MS2 by Gly-Ser linker. And this is an improvement of both parts. If the vaccine is uncontrollable, TMP will be administered that will stabilize DD therefore, binding of MS2 to U2-snRNP that finally inhibits the circuit and stops the transcription in extreme conditions.
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===Characterization Of Mutational Landscape===
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Team:<html><a href="https://2021.igem.org/Team:AFCM-Egypt">AFCM-Egypt 2021</a></html>has characterized & improved this part As shown in <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K3743002">BBa_K3743002</a></html> by A mutational landscape prediction through saturation mutagenesis of MS2 protein and the effect of these mutations on the evolutionary fitness of the protein is tested after generating multiple sequence alignment of the protein sequence and predict mutational landscapes. As shown in the chart, the (W70T) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (L74G) contributed to the lowest evolutionary fitness to MS2. As shown in Figure (1)
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[[File:T--AFCM-EGYPT--MS2-2.PNG|thumb|left|Figure 1.shows the positive fit mutants upon saturation mutagenesis prediction of mutational landscape of Ms2]]
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<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
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Furthermore, Auslander et al [3] also showed that the MS2 protein-box relationship can be designed for use in synthetic biology to create programmable single-cell mammalian biocomputers with simple expression logic, increasing the complexity of a informational processing system without researcher input.
 
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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<p>The MS2 coat-protein was included in our replicon.</p>
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<html><body><img src="https://static.igem.org/mediawiki/2014/thumb/9/9e/MS2CP_Schematic.jpg/800px-MS2CP_Schematic.jpg"/></body></html>
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Latest revision as of 17:12, 20 October 2021

MS2 bacteriophage coat protein


Regulating the expression of a protein can be achieved through repressive elements; we have the MS2 and MS2 hairpin, which regulate the production of RNA Dependent RNA polymerase (RdRp). As RdRp is fused with MS2 through a P2A linker, an indefinite increase in RdRp production is rate-limited by MS2 production. MS2 is able to bind to the MS2 hairpin sequence, also rate-limiting RdRp production, which in turn regulates the whole replicon system. The iGEM community can benefit from a regulation system, in which MS2 can be fused to a protein of interest to regulate its expression. If the fusion protein is non-functional, engineering restriction sites in the P2A linker and site specific ligation reconstitutes RdRp without the MS2 hairpin. The part derives from Auslander et al, 2012, which showed that the MS2 protein-box relationship can be designed for use in synthetic biology to create programmable single-cell mammalian biocomputers with simple expression logic, increasing the complexity of an information processing system without researcher input.

Characterization & Improvement by AFCM-Egypt 2021

Improvement by addition of a Destabilizing domain

Team:AFCM-Egypt 2021has improved this part As shown in BBa_K3743004 by addition of destabilization domains (DD) which shows a fusion protein component that is unstable and destabilizes other proteins, leading to protein degradation. A well-known example of DD is mutated DHFR (E. coli) (DDDHFR), which causes the degradation of proteins in cells. However, if a trimethoprim analog (TMP) is added to bind the destabilizing domain, the fusion protein can stabilize and return the expression to normal levels. This approach allows the regulation of secreted proteins and their biological activity, allowing functional control over proteins of interest in a variety of contexts in mammalian cell cultures, the Plasmodium and Toxoplasma apicomplexans, and in live mice, creating new avenues to open various applications such as cancer therapy and targeted gene delivery. In our part, the destabilizing domain binds to the ms2 coat protein that inhibits it from binding to its riboswitch until trimethoprim analog (TMP) is added which acts as a small molecule inhibitor will free the MS2 after stabilizing DD. Free MS2 inhibits the circuit to be able to control the transcription process in extreme conditions as cytokine storm or unpredictable results. This part represents the final form of the used riboswitch to be controlled by Trimethoprim (TMP) that has a small molecule inhibitor role. Destabilizing domain (DD) is fused to MS2 by Gly-Ser linker. And this is an improvement of both parts. If the vaccine is uncontrollable, TMP will be administered that will stabilize DD therefore, binding of MS2 to U2-snRNP that finally inhibits the circuit and stops the transcription in extreme conditions.

Characterization Of Mutational Landscape

Team:AFCM-Egypt 2021has characterized & improved this part As shown in BBa_K3743002 by A mutational landscape prediction through saturation mutagenesis of MS2 protein and the effect of these mutations on the evolutionary fitness of the protein is tested after generating multiple sequence alignment of the protein sequence and predict mutational landscapes. As shown in the chart, the (W70T) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (L74G) contributed to the lowest evolutionary fitness to MS2. As shown in Figure (1)

Figure 1.shows the positive fit mutants upon saturation mutagenesis prediction of mutational landscape of Ms2















Usage and Biology

The MS2 coat-protein was included in our replicon.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 255
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 255
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 255
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 255
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 255
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 292