Difference between revisions of "Part:BBa K4347012:Design"
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<partinfo>BBa_K4347012 short</partinfo>
 
<partinfo>BBa_K4347012 short</partinfo>
  
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===Design Notes===
 
===Design Notes===
  
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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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This fusion polymerase was an iteration of the re-engineered Bst, incorporating an improved thermally stable version of Bst and thermal stable DNA binding protien Sso7d. An AlphaFold analysis in combination with Pymol was used to predict the structure of this synthetic polymerase. Sso7d was fused via N-terminal to Bst using a flexible (GGGGS)<sub>4</sub> linker.
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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>
 
<u>Bst Mutagenesis</u>
  
 
<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>
 
<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>
 
<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>
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DNA-BP Sso7d was fused to wildtype Bst polymerase by iGEM Fudan in 2021 to improve polymerase processivity in LAMP. We wanted to build upon this creation through fusing Sso7d to an thermally improved Bst polymerase to improve the overall LAMP test for POC applications. DNA-BP Sso7d has been proven to increase Bst homologue Taq polymerases' processivity numerous times in PCR reactions[[Part:BBa_K4347012:Design#References|<sup>[1]</sup>]] [[Part:BBa_K4347012:Design#References|<sup>[2]</sup>]], thus we wanted to see if the same effects would apply to LAMP.
  
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===Design Considerations===
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===Considerations===
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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_K4347012: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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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_K4347012:Design#References|<sup>[3]</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.
  
 
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.
 
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.
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===References===
 
===References===
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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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1. Oscorbin, I. P., Wong, P. F., Boyarskikh, U. A., Khrapov, E. A., &amp; Filipenko, M. L. (2020). The attachment of a dna‐binding sso7d‐like protein improves processivity and resistance to inhibitors of M‐MULV reverse transcriptase. FEBS Letters, 594(24), 4338–4356. https://doi.org/10.1002/1873-3468.13934
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2. Wang, Y. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research, 32(3), 1197–1207. https://doi.org/10.1093/nar/gkh271
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3. 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

Latest revision as of 23:11, 1 October 2022

Bst fusion with Sso7d 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 971
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

This fusion polymerase was an iteration of the re-engineered Bst, incorporating an improved thermally stable version of Bst and thermal stable DNA binding protien Sso7d. An AlphaFold analysis in combination with Pymol was used to predict the structure of this synthetic polymerase. Sso7d was fused via N-terminal to Bst using a flexible (GGGGS)4 linker.

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

DNA-BP Sso7d was fused to wildtype Bst polymerase by iGEM Fudan in 2021 to improve polymerase processivity in LAMP. We wanted to build upon this creation through fusing Sso7d to an thermally improved Bst polymerase to improve the overall LAMP test for POC applications. DNA-BP Sso7d has been proven to increase Bst homologue Taq polymerases' processivity numerous times in PCR reactions[1] [2], thus we wanted to see if the same effects would apply to LAMP.

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[3], 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.

Source

References

1. Oscorbin, I. P., Wong, P. F., Boyarskikh, U. A., Khrapov, E. A., & Filipenko, M. L. (2020). The attachment of a dna‐binding sso7d‐like protein improves processivity and resistance to inhibitors of M‐MULV reverse transcriptase. FEBS Letters, 594(24), 4338–4356. https://doi.org/10.1002/1873-3468.13934

2. Wang, Y. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research, 32(3), 1197–1207. https://doi.org/10.1093/nar/gkh271

3. 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