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_K4347010#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity[[Part:BBa_K4347010#References|<sup>[2]</sup>]]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP.
 +
[[File:Bst2.png|200px|center|thumb|Bst polymerase derived from thermophilic bacterium <em>Geobacillus stearothermophilus</em> used in LAMP modelled on PyMol.]]
 +
 
 +
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_K4347010#References|<sup>[3]</sup>]].
 +
[[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). 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>[1]</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.
+
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>[3]</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.
  
 
[[File:BBa K43470011 bst fusion stability.PNG|400px|center|thumb|Thermal stability of Bst fusion protien computed from YASARA.
 
[[File:BBa K43470011 bst fusion stability.PNG|400px|center|thumb|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 <em>Sulfolobus acidocaldarius</em> and is part of the  7 kDa DNA-binding family[[Part:BBa_K4347011#References|<sup>[2]</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>[3]</sup>]]. 7 kDa DNA-binging protiens have been shown to increase processivity when fused to polymerases such as Taq[[Part:BBa_K4347011#References|<sup>[4]</sup>]].  
+
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>[2]</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>[4]</sup>]]. 7 kDa DNA-binging protiens have been shown to increase processivity when fused to polymerases such as Taq[[Part:BBa_K4347011#References|<sup>[5]</sup>]].  
  
 
[[File:BBa K4347011 bst full fusion.PNG|400px|center|thumb|Fully modified Bst polymerase with fusion protien and point mutations modelled in Pymol.]]
 
[[File:BBa K4347011 bst full fusion.PNG|400px|center|thumb|Fully modified Bst polymerase with fusion protien and point mutations modelled in Pymol.]]
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===References===
 
===References===
 
<br>  
 
<br>  
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1. 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
+
1.
  
 
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
 
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
  
−
3. 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  
+
3. 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
 +
 
 +
 
 +
4. 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  
  
−
4. 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
+
5. 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

Revision as of 20:15, 20 July 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.

Bst polymerase derived from thermophilic bacterium Geobacillus stearothermophilus used in LAMP modelled on PyMol.

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[3]. 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[2]. 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[4]. 7 kDa DNA-binging protiens have been shown to increase processivity when fused to polymerases such as Taq[5].

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


Lab results and pictures


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.

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

3. 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


4. 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

5. 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