Difference between revisions of "Part:BBa K4347007"
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<partinfo>BBa_K4347007 short</partinfo>
 
<partinfo>BBa_K4347007 short</partinfo>
  
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This part is an improvement from Bst Polymerase 1 (large fragment) for E.coli from the 2021 iGEM Fudan team: https://parts.igem.org/Part:BBa_K3790000.  
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This part is an improvement from Bst Polymerase I (large fragment) for E.coli from the 2021 iGEM Fudan team: https://parts.igem.org/Part:BBa_K3790000.  
 
   
 
   
 
__TOC__
 
__TOC__
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===Usage and Biology===
 
===Usage and Biology===
  
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Bst polymerase is a family I DNA polymerase derived from the thermophilic bacterium <em> Geobacillus stearothermophilus.</em> Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability [[Part:BBa_K4347007#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity [[Part:BBa_K4347007#References|<sup>[2]</sup>]]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP.  
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Bst polymerase Large Fragment is a family I DNA polymerase derived from the thermophilic bacterium <em> Geobacillus stearothermophilus.</em> Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability [[Part:BBa_K4347007#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity [[Part:BBa_K4347007#References|<sup>[2]</sup>]]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP.  
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[[File:BBa K4347007-bst taq.PNG|150px|left|thumb|Bst polymerase (green) superimposed with Klentaq fragment (white) from Taq polymerase in Pymol.]]
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[[File:BBa K4347007-bst KF.PNG|150px|right|thumb|Bst polymerase (green) superimposed with Klenow fragment (white) from DNA polymerase I from <em> E.coli</em> in Pymol.]]
  
 
[[File: BBa K4347007--bst.PNG|300.px|center|thumb|Bst polymerase derived from thermophilic bacterium <em> Geobacillus stearothermophilus </em> used in LAMP modelled on PyMol.]]
 
[[File: BBa K4347007--bst.PNG|300.px|center|thumb|Bst polymerase derived from thermophilic bacterium <em> Geobacillus stearothermophilus </em> used in LAMP modelled on PyMol.]]
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Bst polymerase large fragment is structurally homologous to KlenTaq polymerase (large fragment of Taq polymerase used in PCR) [[Part:BBa_K4347007#References|<sup>[3]</sup>]] and Klenow fragment (large fragment of DNA polymerase I in E. coli) [[Part:BBa_K4347007#References|<sup>[4]</sup>]].
  
 
===Enhanced Thermal stability===
 
===Enhanced Thermal stability===
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Talk about the point mutations.  
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The origional codon optimized Bst polymerase (Part BBa_K3790000) was improved upon to further enhance the polymerases thermal stability such that it can carry out LAMP at a higher reaction temperature. Three point mutations were made in the polymerase thumb domain: K549W, K582L and Q584L. The increased thermal stability from these point mutations was validated using a protien simulation program called YASARA. The overall change in Gibbs free energy of wild-type Bst was calculated to be -150.13 kcal/mol, and the overall stability of the mutated Bst was calculated to be -151.81 kcal/mol thus indicative of a more thermally stable protein.
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Pictures of lab experiments
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[[File:BBa K4347007 stability point muts.PNG|700.px|center|thumb|Thermal stability of mutated Bst polymerase computed from YASARA.]]
  
 
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===References===
 
===References===
 
<br>
 
<br>
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1. https://www.tandfonline.com/doi/full/10.1080/10826068.2022.2095573?src=
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1. Ignatov, K. B., Barsova, E. V., Fradkov, A. F., Blagodatskikh, K. A., Kramarova, T. V., & Kramarov, V. M. (2014). A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques, 57(2), 81–87. https://doi.org/10.2144/000114198
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2. Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H (1996). Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. (5-6):185-95. PMID: 8740835
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3. Milligan, J. N., Shroff, R., Garry, D. J., &amp; Ellington, A. D. (2018). Evolution of a thermophilic strand-displacing polymerase using high-temperature isothermal compartmentalized self-replication. Biochemistry, 57(31), 4607–4619. https://doi.org/10.1021/acs.biochem.8b00200
  
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2. https://pubmed.ncbi.nlm.nih.gov/8740835/
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4.Kiefer, J. R., Mao, C., Hansen, C. J., Basehore, S. L., Hogrefe, H. H., Braman, J. C., &amp; Beese, L. S. (1997, February). Crystal structure of a Thermostable Bacillus DNA polymerase I large Fragment at 2.1A Resolution. ResearchGate. Retrieved July 11, 2022, from https://www.researchgate.net/publication/14191850_Crystal_Structure_of_a_Thermostable_Bacillus_DNA_Polymerase_I_Large_Fragment_at_21_A_Resolution
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Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H. Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Genet Anal. 1996 Mar;12(5-6):185-95. PMID: 8740835
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Latest revision as of 17:06, 20 July 2022

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

This part is an improvement from Bst Polymerase I (large fragment) for E.coli from the 2021 iGEM Fudan team: https://parts.igem.org/Part:BBa_K3790000.

Usage and Biology

Bst polymerase Large Fragment is a family I DNA polymerase derived from the thermophilic bacterium Geobacillus stearothermophilus. Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability [1]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity [2]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP.

Bst polymerase (green) superimposed with Klentaq fragment (white) from Taq polymerase in Pymol.
Bst polymerase (green) superimposed with Klenow fragment (white) from DNA polymerase I from E.coli in Pymol.
Bst polymerase derived from thermophilic bacterium Geobacillus stearothermophilus used in LAMP modelled on PyMol.

Bst polymerase large fragment is structurally homologous to KlenTaq polymerase (large fragment of Taq polymerase used in PCR) [3] and Klenow fragment (large fragment of DNA polymerase I in E. coli) [4].

Enhanced Thermal stability

The origional codon optimized Bst polymerase (Part BBa_K3790000) was improved upon to further enhance the polymerases thermal stability such that it can carry out LAMP at a higher reaction temperature. Three point mutations were made in the polymerase thumb domain: K549W, K582L and Q584L. The increased thermal stability from these point mutations was validated using a protien simulation program called YASARA. The overall change in Gibbs free energy of wild-type Bst was calculated to be -150.13 kcal/mol, and the overall stability of the mutated Bst was calculated to be -151.81 kcal/mol thus indicative of a more thermally stable protein.

Thermal stability of mutated Bst polymerase computed from YASARA.


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 766
  • 1000
    COMPATIBLE WITH RFC[1000]


References


1. Ignatov, K. B., Barsova, E. V., Fradkov, A. F., Blagodatskikh, K. A., Kramarova, T. V., & Kramarov, V. M. (2014). A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques, 57(2), 81–87. https://doi.org/10.2144/000114198

2. Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H (1996). Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. (5-6):185-95. PMID: 8740835

3. Milligan, J. N., Shroff, R., Garry, D. J., & Ellington, A. D. (2018). Evolution of a thermophilic strand-displacing polymerase using high-temperature isothermal compartmentalized self-replication. Biochemistry, 57(31), 4607–4619. https://doi.org/10.1021/acs.biochem.8b00200

4.Kiefer, J. R., Mao, C., Hansen, C. J., Basehore, S. L., Hogrefe, H. H., Braman, J. C., & Beese, L. S. (1997, February). Crystal structure of a Thermostable Bacillus DNA polymerase I large Fragment at 2.1A Resolution. ResearchGate. Retrieved July 11, 2022, from https://www.researchgate.net/publication/14191850_Crystal_Structure_of_a_Thermostable_Bacillus_DNA_Polymerase_I_Large_Fragment_at_21_A_Resolution