Difference between revisions of "Part:BBa K4347011"

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===Usage and Biology===
 
===Usage and Biology===
  
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Usage of bst and Sac7e in biology. Can summarize what was written for "bst with point mutations" and "sac7e" pages. This bst version DOES include the point mutations
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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_K4347011#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity[[Part:BBa_K4347011#References|<sup>[2]</sup>]]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP. Three point mutations were introduced at positions K549W, K582L,  and Q584L in the thumb domain to improve polymerase thermal stability.  
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[[File:BBa K4347007 bst point mut.PNG|300px|center|thumb|Full Bst structure with point mutations (orange) in thumb domain.]]
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Sac7e is part of the 7 kDa DNA-binding family and is a highly thermostable and pH resistant protien that aids in the binding of double stranded DNA. Sac7e is thermally stable to 85.5°C and compared to other similar proteins, Sac7e showed the highest affinity for dsDNA (KD = 11 μM), with binding sites ~ 6-8 bases per protein[[Part:BBa_K4347011#References|<sup>[3]</sup>]].
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[[File:BBa K4347006 sac7e.PNG|200px|center|thumb|DNA binding protien "Sac7e" modelled in Pymol.]]
  
 
===A more thermally stable and processive polymerase===
 
===A more thermally stable and processive polymerase===
  
−
This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006).
+
This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006). The modified Bst polymerase contains three point mutations in the polymerase thumb domain: K549W, K582L and Q584L, which have been proven to improve thermal stability in Bst homologue Taq polymerase[[Part:BBa_K4347011#References|<sup>[4]</sup>]]. 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 -152.03 kcal/mol thus indicative of a more thermally stable protein.
  
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Small summary on the new polymerase, combining parts https://parts.igem.org/Part:BBa_K4347006 and https://parts.igem.org/Part:BBa_K4347007#
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[[File:BBa K43470011 bst fusion stability.PNG|400px|center|thumb|Thermal stability of Bst fusion protien computed from YASARA.
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[[File:BBa K43470011 bst fusion stability.PNG|500px|center|thumb|Thermal stability of Bst fusion protien computed from YASARA.
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]]
 
]]
  
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Along with an increased thermal stability, the mutated polymerase was fused to a DNA binding protien Sac7e to increase polymerase processivity during the LAMP reaction. Sac7e is isolated from thermoacidophilic archaeon <em>Sulfolobus acidocaldarius</em> and is part of the  7 kDa DNA-binding family[[Part:BBa_K4347011#References|<sup>[3]</sup>]]. Sac7e binds to DNA without a strong sequence preference. In complex with DNA, a small beta-barrel is capped by anamphiphilic C-terminal a-helix. The triple-stranded beta-sheet is placed across the DNA minor groove with the intercalation of the Val26 and Met29 side-chains into DNA base-pairs, causing a sharp kink in the DNA duplex[[Part:BBa_K4347011#References|<sup>[5]</sup>]]. 7 kDa DNA-binging protiens have been shown to increase processivity when fused to polymerases such as Taq[[Part:BBa_K4347011#References|<sup>[6]</sup>]].
  
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Lab results and pictures
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[[File:BBa K4347011 bst full fusion.PNG|350px|center|thumb|Fully modified Bst polymerase with Sac7e fusion and point mutations modelled in Pymol.]]
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 +
===Results===
 +
Our team was able to successfully express this protien in E.coli cells and purified through nickel chromatography.
 +
[[File:BBa K4347011 expression.PNG|250px|center|thumb|SDS-PAGE gel for Sac7e fusion protien after expression and purification. Crude, supernatant, pellet, and elution samples for both uninduced and IPTG-induced proteins are displayed.]]
  
 
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===References===
 
===References===
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<br>
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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
 +
 +
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. 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
 +
 +
4. Xi, L. (2009, December 23). WO2009155464A2 - mutated and chemically modified thermally stable DNA polymerases. Google Patents. Retrieved July 12, 2022, from https://patents.google.com/patent/WO2009155464A2/en
 +
 +
5. Su, S., Gao, Y.-G., Robinson, H., Liaw, Y.-C., Edmondson, S. P., Shriver, J. W., &amp; Wang, A. H.-J. (2000). Crystal structures of the chromosomal proteins SSO7D/sac7d bound to DNA containing T-G mismatched base-pairs. Journal of Molecular Biology, 303(3), 395–403. https://doi.org/10.1006/jmbi.2000.4112
 +
 +
6. 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

Latest revision as of 03:31, 2 October 2022

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

This fusion protien was designed by linking the N-terminus of a modified Bst polymerase with thermostable DNA binding protien Sac7e using a flexible (GGGGS)4 linker to increase polymerase thermostability and processivity in LAMP reaction.

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. Three point mutations were introduced at positions K549W, K582L, and Q584L in the thumb domain to improve polymerase thermal stability.

Full Bst structure with point mutations (orange) in thumb domain.

Sac7e is part of the 7 kDa DNA-binding family and is a highly thermostable and pH resistant protien that aids in the binding of double stranded DNA. Sac7e is thermally stable to 85.5°C and compared to other similar proteins, Sac7e showed the highest affinity for dsDNA (KD = 11 μM), with binding sites ~ 6-8 bases per protein[3].

DNA binding protien "Sac7e" modelled in Pymol.

A more thermally stable and processive polymerase

This final iteration of the new polymerase is an improvement of our previous part; BBa_K4347010, as it is a combination of our more thermally stable polymerase (BBa_K4347007) fused with DNA binding protien Sac7e (BBa_K4347006). The modified Bst polymerase contains three point mutations in the polymerase thumb domain: K549W, K582L and Q584L, which have been proven to improve thermal stability in Bst homologue Taq polymerase[4]. 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 -152.03 kcal/mol thus indicative of a more thermally stable protein.

Thermal stability of Bst fusion protien computed from YASARA.

Along with an increased thermal stability, the mutated polymerase was fused to a DNA binding protien Sac7e to increase polymerase processivity during the LAMP reaction. Sac7e is isolated from thermoacidophilic archaeon Sulfolobus acidocaldarius and is part of the 7 kDa DNA-binding family[3]. Sac7e binds to DNA without a strong sequence preference. In complex with DNA, a small beta-barrel is capped by anamphiphilic C-terminal a-helix. The triple-stranded beta-sheet is placed across the DNA minor groove with the intercalation of the Val26 and Met29 side-chains into DNA base-pairs, causing a sharp kink in the DNA duplex[5]. 7 kDa DNA-binging protiens have been shown to increase processivity when fused to polymerases such as Taq[6].

Fully modified Bst polymerase with Sac7e fusion and point mutations modelled in Pymol.

Results

Our team was able to successfully express this protien in E.coli cells and purified through nickel chromatography.

SDS-PAGE gel for Sac7e fusion protien after expression and purification. Crude, supernatant, pellet, and elution samples for both uninduced and IPTG-induced proteins are displayed.


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]


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. 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

4. Xi, L. (2009, December 23). WO2009155464A2 - mutated and chemically modified thermally stable DNA polymerases. Google Patents. Retrieved July 12, 2022, from https://patents.google.com/patent/WO2009155464A2/en

5. Su, S., Gao, Y.-G., Robinson, H., Liaw, Y.-C., Edmondson, S. P., Shriver, J. W., & Wang, A. H.-J. (2000). Crystal structures of the chromosomal proteins SSO7D/sac7d bound to DNA containing T-G mismatched base-pairs. Journal of Molecular Biology, 303(3), 395–403. https://doi.org/10.1006/jmbi.2000.4112

6. 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