Composite

Part:BBa_K4347012

Designed by: Victor Di Donato, Nicoletta de Maat   Group: iGEM22_Queens_Canada   (2022-10-01)
Revision as of 03:30, 2 October 2022 by Victor5688 (Talk | contribs) (→‎Results)

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 gels 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. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685558/

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