Difference between revisions of "Part:BBa K4347012"
(→‎References)
 
(6 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
partinfo>BBa_K4347011 short</partinfo>
+
<partinfo>BBa_K4347012 short</partinfo>
 
__TOC__
 
__TOC__
−
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.
+
This fusion protien was designed by linking the N-terminus of a modified Bst polymerase with thermostable DNA binding protien Sso7d using a flexible (GGGGS)<sub>4</sub> linker to increase polymerase thermostability and processivity in LAMP reaction.
  
 
===Usage and Biology===
 
===Usage and Biology===
Line 8: Line 8:
 
[[File:BBa K4347007 bst point mut.PNG|300px|center|thumb|Full Bst structure with point mutations (orange) in thumb domain.]]
 
[[File:BBa K4347007 bst point mut.PNG|300px|center|thumb|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[[Part:BBa_K4347012#References|<sup>[3]</sup>]].
+
Sso7d 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.[[Part:BBa_K4347012#References|<sup>[3]</sup>]]. Sso7d is thermally stable to 100°C and has been shown to promote annealing of complementary DNA strands, induces negative supercoiling and chaperones the disassembly and renaturation of protien aggregates in an ATP hydrolysis-dependent manner. [[Part:BBa_K4347012#References|<sup>[4]</sup>]]
 
[[File:BBa K4347012 sso7d.PNG|200px|center|thumb|DNA binding protien "Sac7e" modelled in Pymol.]]
 
[[File:BBa K4347012 sso7d.PNG|200px|center|thumb|DNA binding protien "Sac7e" modelled in Pymol.]]
  
 +
===Results===
 +
We modelled this fusion polymerase using FoldX and Pymol to ensure the structures did not interfere.
 +
[[File:BBa K4347012 sso7d fusion.PNG|350px|center|thumb|Fully modified Bst polymerase with Sso7d fusion and point mutations modelled in Pymol.]]
  
−
 
+
−
<!--Lab results and pictures-->
+
Our team was successfully able to express this protien in E.coli and purify through nickel chromatography. We also confirmed our result through a Western Blot.
−
 
+
[[File:BBa K4347012 expression Sso7d.PNG|250px|center|thumb|SDS-PAGE gel for Sso7d fusion protien after expression and purification. Crude, supernatant, pellet, and elution samples for both uninduced and IPTG-induced proteins are displayed. Band at 74.6 kDa indicates successful purification.]]
  
 
<!-- -->
 
<!-- -->
Line 33: Line 36:
 
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
 
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
+
4. Guagliardi, A., Cerchia, L., &amp; Rossi, M. (2002). The SSO7D protein ofsulfolobus solfataricus: In vitro relationship among different activities. Archaea, 1(2), 87–93. https://doi.org/10.1155/2002/313147
−
 
+
−
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 03:42, 2 October 2022

Bst fusion with Sso7d 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 Sso7d 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.

Sso7d 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.[3]. Sso7d is thermally stable to 100°C and has been shown to promote annealing of complementary DNA strands, induces negative supercoiling and chaperones the disassembly and renaturation of protien aggregates in an ATP hydrolysis-dependent manner. [4]

DNA binding protien "Sac7e" modelled in Pymol.

Results

We modelled this fusion polymerase using FoldX and Pymol to ensure the structures did not interfere.

Fully modified Bst polymerase with Sso7d fusion and point mutations modelled in Pymol.


Our team was successfully able to express this protien in E.coli and purify through nickel chromatography. We also confirmed our result through a Western Blot.

SDS-PAGE gel for Sso7d fusion protien after expression and purification. Crude, supernatant, pellet, and elution samples for both uninduced and IPTG-induced proteins are displayed. Band at 74.6 kDa indicates successful purification.


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. Guagliardi, A., Cerchia, L., & Rossi, M. (2002). The SSO7D protein ofsulfolobus solfataricus: In vitro relationship among different activities. Archaea, 1(2), 87–93. https://doi.org/10.1155/2002/313147