Difference between revisions of "Part:BBa K4347011:Design"
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===Design Notes===
 
===Design Notes===
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Research that lead to choosing sac7e as ssb protien.  
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Alpha fold analysis
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This fusion polymerase was the final iteration of the re-engineered Bst, incorporating an improved thermally stable version of Bst and thermal stable DNA binding protien Sac7e. An AlphaFold analysis in combination with Pymol was used to predict the structure of this synthetic polymerase. Sac7e was fused via N-terminal to Bst using a flexible (GGGGS)<sub>4</sub> linker.
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Yasara analysis
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[[File:BBa K4347011 bst full fusion.PNG|350px|center|thumb|Fully modified Bst polymerase with fusion protien and point mutations modelled in Pymol.]]
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<u>Bst Mutagenesis</u>
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<b>For more information and a complete overview on how the mutated polymerase was designed please view: https://parts.igem.org/Part:BBa_K4347007:Design </b>
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<u>DNA Binding Protien</u>
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<b>For more information on why Sac7e was selected please view the considerations section on:https://parts.igem.org/Part:BBa_K4347010:Design#Considerations </b>
  
 
===Considerations===
 
===Considerations===
  
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Section can be summary from https://parts.igem.org/Part:BBa_K4347007# and https://parts.igem.org/Part:BBa_K4347010.
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Point mutations were made for thermal stability to account for fluctuations in the portable heating device, as temperature fluctuations typically oscillate about the desired set point temperature when using electronic circuits. The basis of our research was based off of Taq polymerase, which is a structural homologue to Bst. In a study by Raghunathan & Marx[[Part:BBa_K4347011:Design#References|<sup>[1]</sup>]], it was found that only 25% of the point mutations made in the fingers domain of Taq resulted in a PCR active polymerase whereas over 70% and 60% of the mutations in the thumb and palm domains resulted in a PCR active polymerase. Due to the sequence similarity of Bst and Taq, these inactive mutations would likely have the same effect on Bst.
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-Considerations with point mutations.  
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- Previous work that had already been done, heating mechanism of our device (might be more than 65 bc its a kettle based device), how fusion would interfere bst structure eg, alpha fold with phi29.  
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The purpose of the DNA binding protien is to enhance processivity of the polymerase to result in more amplification product to yield a greater endpoint signal in our indicator via magnesium ion depletion to LAMP byproduct pyrophosphate. Sac7e showed the highest affinity for double stranded DNA when compared to homologues in its family and is thermally stable up to Sac7e 85.5°C[[Part:BBa_K4347011:Design#References|<sup>[2]</sup>]]. Through our YASARA analysis, its fusion confers extra thermal stability to the modified Bst (part BBa_K4347007) by 0.24 kcal/mol.
  
 
===Source===
 
===Source===
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* Sac7e: https://parts.igem.org/Part:BBa_K4347006
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Bst: https://parts.igem.org/Part:BBa_K3790000
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* (GGGGS)<sub>4</sub> linker: https://parts.igem.org/Part:BBa_K3117004
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Sac7e: https://parts.igem.org/Part:BBa_K4347006
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* Bst: https://parts.igem.org/Part:BBa_K4347010
  
 
===References===
 
===References===
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<br>
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1. Raghunathan, G., & Marx, A. (2019, January 24). Identification of thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Nature News. Retrieved July 12, 2022, from https://www.nature.com/articles/s41598-018-37233-y#Fig6
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2. Kalichuk, V., Béhar, G., Renodon-Cornière, A., Danovski, G., Obal, G., Barbet, J., Mouratou, B., & Pecorari, F. (2016). The archaeal “7 KDA DNA-binding” proteins: Extended characterization of an old gifted family. Scientific Reports, 6(1). https://doi.org/10.1038/srep37274

Latest revision as of 02:00, 30 July 2022


Bst fusion with Sac7e and point mutations for enhanced thermal stability codon optimized for E.coli


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 5
    Illegal XhoI site found at 209
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1015
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

This fusion polymerase was the final iteration of the re-engineered Bst, incorporating an improved thermally stable version of Bst and thermal stable DNA binding protien Sac7e. An AlphaFold analysis in combination with Pymol was used to predict the structure of this synthetic polymerase. Sac7e was fused via N-terminal to Bst using a flexible (GGGGS)4 linker.

Fully modified Bst polymerase with fusion protien and point mutations modelled in Pymol.


Bst Mutagenesis

For more information and a complete overview on how the mutated polymerase was designed please view: https://parts.igem.org/Part:BBa_K4347007:Design


DNA Binding Protien

For more information on why Sac7e was selected please view the considerations section on:https://parts.igem.org/Part:BBa_K4347010:Design#Considerations

Considerations

Point mutations were made for thermal stability to account for fluctuations in the portable heating device, as temperature fluctuations typically oscillate about the desired set point temperature when using electronic circuits. The basis of our research was based off of Taq polymerase, which is a structural homologue to Bst. In a study by Raghunathan & Marx[1], it was found that only 25% of the point mutations made in the fingers domain of Taq resulted in a PCR active polymerase whereas over 70% and 60% of the mutations in the thumb and palm domains resulted in a PCR active polymerase. Due to the sequence similarity of Bst and Taq, these inactive mutations would likely have the same effect on Bst.

The purpose of the DNA binding protien is to enhance processivity of the polymerase to result in more amplification product to yield a greater endpoint signal in our indicator via magnesium ion depletion to LAMP byproduct pyrophosphate. Sac7e showed the highest affinity for double stranded DNA when compared to homologues in its family and is thermally stable up to Sac7e 85.5°C[2]. Through our YASARA analysis, its fusion confers extra thermal stability to the modified Bst (part BBa_K4347007) by 0.24 kcal/mol.

Source

References


1. Raghunathan, G., & Marx, A. (2019, January 24). Identification of thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Nature News. Retrieved July 12, 2022, from https://www.nature.com/articles/s41598-018-37233-y#Fig6

2. Kalichuk, V., Béhar, G., Renodon-Cornière, A., Danovski, G., Obal, G., Barbet, J., Mouratou, B., & Pecorari, F. (2016). The archaeal “7 KDA DNA-binding” proteins: Extended characterization of an old gifted family. Scientific Reports, 6(1). https://doi.org/10.1038/srep37274