Difference between revisions of "Part:BBa K4347012"

 
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partinfo>BBa_K4347011 short</partinfo>
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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)<sub>4</sub> linker to increase polymerase thermostability and processivity in LAMP reaction.
  
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__NOTOC__
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===Usage and Biology===
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<partinfo>BBa_K4347012 short</partinfo>
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Bst fusion with Sso7d and point mutations for enhanced thermal stability codon optimized for E.coli
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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_K4347012#References|<sup>[1]</sup>]]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity[[Part:BBa_K4347012#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_K4347012#References|<sup>[3]</sup>]].
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[[File:BBa K4347006 sac7e.PNG|200px|center|thumb|DNA binding protien "Sac7e" modelled in Pymol.]]
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4347011 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4347012 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4347012 parameters</partinfo>
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<partinfo>BBa_K4347011 parameters</partinfo>
 
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===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
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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. 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
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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
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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
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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

Revision as of 02:31, 2 October 2022

partinfo>BBa_K4347011 short</partinfo>

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.




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